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12.6: New Page - Biology
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12.6: New Page - Biology
A GUIDE TO WRITING SCIENTIFIC PAPERS
Scientific experiments are demanding, exciting endeavors, but, to have an impact, results must be communicated to others. A research paper is a method of communication, an attempt to tell others about some specific data that you have gathered and what you think those data mean in the context of your research. The "rules" of writing a scientific paper are rigid and are different from those that apply when you write an English theme or a library research paper. For clear communication, the paper obviously requires proper usage of the English language and this will be considered in evaluating your reports. Scientific papers must be written clearly and concisely so that readers with backgrounds similar to yours can understand easily what you have done and how you have done it should they want to repeat or extend your work. When writing papers for the biology department, you can assume that your audience will be readers like yourselves with similar knowledge.
Although scientific journals differ somewhat in their specific requirements, a general format that would be acceptable for most biological journals is:
The section headings (Abstract, Introduction, etc.) should be centered and the body of each section should follow immediately below the heading. Do not begin each section on a new page. If one section ends part of the way down the page, the next section heading follows immediately on the same page.
One important general rule to keep in mind is that a scientific paper is a report about something that has been done in the past. Most of the paper should be written in the PAST TENSE (was, were). The present tense (is, are) is used when stating generalizations or conclusions. The present tense is most often used in the Introduction, Discussion and Conclusion sections of papers. The paper should read as a narrative in which the author describes what was done and what results were obtained from that work.
Every scientific paper must have a self-explanatory title. By reading the title, the work being reported should be clear to the reader without having to read the paper itself. The title, "A Biology Lab Report", tells the reader nothing. An example of a good, self-explanatory title would be: "The Effects of Light and Temperature on the Growth of Populations of the Bacterium, Escherichia coli ". This title reports exactly what the researcher has done by stating three things:
1. The environmental factors that were manipulated (light, temperature).
2. The parameter that was measured (growth).
3. The specific organism that was studied (the bacterium, Escherichia coli).
If the title had been only "Effects of Light and Temperature on Escherichia coli ", the reader would have to guess which parameters were measured. (That is, were the effects on reproduction, survival, dry weight or something else?) If the title had been "Effect of Environmental Factors on Growth of Escherichia coli ", the reader would not know which environmental factors were manipulated. If the title had been "Effects of Light and Temperature on the Growth of an Organism", then the reader would not know which organism was studied. In any of the above cases, the reader would be forced to read more of the paper to understand what the researcher had done.
Exceptions do occur: If several factors were manipulated, all of them do not have to be listed. Instead, "Effects of Several Environmental Factors on Growth of Populations ofEscherichia coli " (if more than two or three factors were manipulated) would be appropriate. The same applies if more than two or three organisms were studied. For example, "Effects of Light and Temperature on the Growth of Four Species of Bacteria" would be correct. The researcher would then include the names of the bacteria in the Materials and Methods section of the paper.
The abstract section in a scientific paper is a concise digest of the content of the paper. An abstract is more than a summary. A summary is a brief restatement of preceding text that is intended to orient a reader who has studied the preceding text. An abstract is intended to be self-explanatory without reference to the paper, but is not a substitute for the paper.
The abstract should present, in about 250 words, the purpose of the paper, general materials and methods (including, if any, the scientific and common names of organisms), summarized results, and the major conclusions. Do not include any information that is not contained in the body of the paper. Exclude detailed descriptions of organisms, materials and methods. Tables or figures, references to tables or figures, or references to literature cited usually are not included in this section. The abstract is usually written last. An easy way to write the abstract is to extract the most important points from each section of the paper and then use those points to construct a brief description of your study.
The Introduction is the statement of the problem that you investigated. It should give readers enough information to appreciate your specific objectives within a larger theoretical framework. After placing your work in a broader context, you should state the specific question(s) to be answered. This section may also include background information about the problem such as a summary of any research that has been done on the problem in the past and how the present experiment will help to clarify or expand the knowledge in this general area. All background information gathered from other sources must, of course, be appropriately cited. (Proper citation of references will be described later.)
A helpful strategy in this section is to go from the general, theoretical framework to your specific question. However, do not make the Introduction too broad. Remember that you are writing for classmates who have knowledge similar to yours. Present only the most relevant ideas and get quickly to the point of the paper. For examples, see the Appendix.
This section explains how and, where relevant, when the experiment was done. The researcher describes the experimental design, the apparatus, methods of gathering data and type of control. If any work was done in a natural habitat, the worker describes the study area, states its location and explains when the work was done. If specimens were collected for study, where and when that material was collected are stated. The general rule to remember is that the Materials and Methods section should be detailed and clear enough so that any reader knowledgeable in basic scientific techniques could duplicate the study if she/he wished to do so. For examples, see the Appendix.
DO NOT write this section as though it were directions in a laboratory exercise book. Instead of writing:
First pour agar into six petri plates. Then inoculate the plates with the bacteria. Then put the plates into the incubator . . .
Simply describe how the experiment was done:
Six petri plates were prepared with agar and inoculated with the bacteria. The plates were incubated for ten hours.
Also, DO NOT LIST the equipment used in the experiment. The materials that were used in the research are simply mentioned in the narrative as the experimental procedure is described in detail. If well-known methods were used without changes, simply name the methods (e.g., standard microscopic techniques standard spectrophotometric techniques). If modified standard techniques were used, describe the changes.
Here the researcher presents summarized data for inspection using narrative text and, where appropriate, tables and figures to display summarized data. Only the results are presented. No interpretation of the data or conclusions about what the data might mean are given in this section. Data assembled in tables and/or figures should supplement the text and present the data in an easily understandable form. Do not present raw data! If tables and/or figures are used, they must be accompanied by narrative text. Do not repeat extensively in the text the data you have presented in tables and figures. But, do not restrict yourself to passing comments either. (For example, only stating that "Results are shown in Table 1." is not appropriate.) The text describes the data presented in the tables and figures and calls attention to the important data that the researcher will discuss in the Discussion section and will use to support Conclusions. (Rules to follow when constructing and presenting figures and tables are presented in a later section of this guide.)
Here, the researcher interprets the data in terms of any patterns that were observed, any relationships among experimental variables that are important and any correlations between variables that are discernible. The author should include any explanations of how the results differed from those hypothesized, or how the results were either different from or similar to those of any related experiments performed by other researchers. Remember that experiments do not always need to show major differences or trends to be important. "Negative" results also need to be explained and may represent something important--perhaps a new or changed focus for your research.
A useful strategy in discussing your experiment is to relate your specific results back to the broad theoretical context presented in the Introduction. Since your Introduction went from the general to a specific question, going from the specific back to the general will help to tie your ideas and arguments together.
This section simply states what the researcher thinks the data mean, and, as such, should relate directly back to the problem/question stated in the introduction. This section should not offer any reasons for those particular conclusions--these should have been presented in the Discussion section. By looking at only the Introduction and Conclusions sections, a reader should have a good idea of what the researcher has investigated and discovered even though the specific details of how the work was done would not be known.
In this section you should give credit to people who have helped you with the research or with writing the paper. If your work has been supported by a grant, you would also give credit for that in this section.
This section lists, in alphabetical order by author, all published information that was referred to anywhere in the text of the paper. It provides the readers with the information needed should they want to refer to the original literature on the general problem. Note that the Literature Cited section includes only those references that were actually mentioned (cited) in the paper. Any other information that the researcher may have read about the problem but did not mention in the paper is not included in this section. This is why the section is called "Literature Cited" instead of "References" or "Bibliography".
The system of citing reference material in scientific journals varies with the particular journal. The method that you will follow is the "author-date" system. Listed below are several examples of how citations should be presented in the text of your paper. The name(s) of the author(s) and year of publication are included in the body of the text. Sentence structure determines the placement of the parentheses.
One author: 'Scott's (1990) model fails to . ' or 'The stream model (Scott 1990) is . '
Two authors: 'Libby and Libby (1991) show. ' or 'Previous moose migration studies (Libby and Libby 1991). '
Three or more authors: 'Roche et al. (1991) reported that . ' or 'During April, moose sightings increased over those in a previous study (Roche et al. 1991) . '
Entries in the Literature Cited section are listed alphabetically by author(s) and chronologically for papers by the same author(s). The following citations illustrate the details of punctuation and order of information for a journal article, book, Internet source, and your laboratory packet.
Schneider, M.J., Troxler, R.F. and Voth, P.D. 1967. Occurrence of indoleacetic acid in the bryophytes. Bot. Gaz. 28(3): 174-179.
Stebbins, G.L. 1977. Processes of Organic Evolution. Prentice-Hall, New Jersey. 269 pp.
MSW Scientific Names: Microtus ochrogaster. Online. Smithsonian Institution. Available: http://www.nmnh.si.edu/cgi-bin/wdb/msw/names/query/22128. updated August 8, 1996 [accessed 8/10/98]
Colby Biology Department. 1998. Salt Tolerance in Phaseolus vulgaris. In: Introduction to Biology: Organismal Biology. Waterville, ME: Colby Custom Publishing
Generally, most references will be to the primary literature (i.e., journal articles) and, to a lesser extent, books. Popular literature and the Internet should be used sparingly and with caution. Other sources such as book chapters and pamphlets typically have their own specific citation formats. If necessary, be sure to find out what these formats are and use them appropriately.
For a much more detailed discussion about writing scientific papers, consult: CBE Style Manual Committee. 1983. CBE Style Manual: A Guide for Authors, Editors and Publishers in the Biological Sciences. 5th Edition, revised and expanded. Council of Biology Editors, Inc., Bethesda, Maryland.
This guide is based on a paper by Gubanich, A.A. 1977. Writing the scientific paper in the investigative lab. Amer. Biol. Teacher, 39(1): 27-34.
Examples from the scientific literature that illustrate material in various sections of a scientific paper.
A. Excerpted from: Hasegawa, K., Sakoda, M. and J. Bruinsma. 1989. Revision of the theory of phototropism in plants: a new interpretation of a classical experiment. Planta 178:540-544.
Went's classical experiment on the diffusion of auxin activity from unilaterally illuminated oat coleoptile tips (Went 1928), was repeated as precisely as possible. In agreement with Went's data with the Avena curvature assay, the agar blocks from the illuminated side of oat (Avena sativa L. cv. Victory) coleoptile tips had, on the average, 38% of the auxin activity of those from the shaded side. However, determination of the absolute amounts of indole-3-acetic acid (IAA) in the agar blocks, using a physicochemical assay following purification, showed that the IAA was evenly distributed in the blocks from the illuminated and shaded sides. In the blocks from the shaded and dark-control halves the amounts of IAA were 2.5 times higher than the auxin activity measured by the Avena curvature test, and in those from the illuminated half even 7 times higher. Chromatography of the diffusates prior to the Avena curvature test demonstrated that the amounts of two growth inhibitors, especially of the more polar one, were significantly higher in the agar blocks from the illuminated side than in those from the shaded side and the dark control. These results show that the basic experiment from which the Cholodny-Went theory was derived does not justify this theory. The data rather indicate that phototropism is caused by the light-induced, local accumulation of growth inhibitors against a background of even auxin distribution, the diffusion of auxin being unaffected.
B. Excerpted from: Farmer, E.E. and Ryan, C.A. 1990. Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Proc. Natl. Acad. Sci. 87: 7713-7716.
Inducible defensive responses in plants are known to be activated locally and systematically by signaling molecules that are produced at sites of pathogen or insect attacks, but only one chemical signal, ethylene, is known to travel through the atmosphere to activate plant defensive genes. Methyl jasmonate, a common plant secondary compound, when applied to surfaces of tomato plants, induces the synthesis of defensive proteinase inhibitor proteins in the treated plants and in nearby plants as well. The presence of methyl jasmonate in the atmosphere of chambers containing plants from three species of two families, Solanaceae and Fabaceae, results in the accumulation of proteinase inhibitors in leaves of all three species. When sagebrush, Artemesia tridentata, a plant shown to possess methyl jasmonate in leaf surface structures, is incubated in chambers with tomato plants, proteinase inhibitor accumulation is induced in the tomato leaves, demonstrating that interplant communication can occur from leaves of one species of plant to leaves of another species to activate the expression of defensive genes.
A. Excerpted from: Shukla, A. and Sawhney, V.K. 1992. Cytokinins in a genic male sterile line of Brassica napus. Physiol. Plant. 85:23-29.
The failure or inability of an individual to produce functional gametes under a given set of environmental conditions is known as sterility. Male sterility in plants is generally associated with the lack of production of viable pollen however its expression can vary (Frankel and Galun 1977, Kaul 1988). In any event, male sterility is of fundamental importance in the production of hybrid seeds and in breeding programs.
Plant growth substances, both exogenously applied and endogenous, have often been implicated in the regulation of male sterility in several plant species (Frankel and Galun 1977, Kaul 1988). Cytokinins, gibberellins, auxins and abscisic acid, as well as polyamines, are all known to affect pollen and stamen development in a number of species (e.g., Sawhney 1974, Ahokas 1982, Saini and Aspinall 1982, Rastogi and Sawhney 1990, Nakajima et al. 1991, Singh et al. 1992).
[Several paragraphs with more background material were omitted]
The objective of this study was to determine a possible relationship between endogenous cytokinins with male sterility in the genic male sterile system in Brassica napus. Thus, an analysis of a number of cytokinins in various organs of the wild type and genic male sterile plants was conducted.
B. Excerpted from: Reader, R.J. and Beisner, B.E. 1991. Species-dependent effects of seed predation and ground cover on seedling emergence of old-field forbs. Am. Midl. Nat. 126: 279-286.
A major goal of plant ecology is to explain spatial variation in a species frequency of occurrence. Spatial variation in seed predation may contribute to spatial variation in plant frequency by reducing seed supply sufficiently to limit seedling emergence more at one location than another (Louda 1982, Anderson 1989). Spatial variation in seed predation is well documented (e.g., Janzen 1971, 1975, Bertness et al. 1987 Smith 1987), but few investigators tested whether differential seed predation resulted in differential seedling emergence (e.g., Louda 1982, 1983). Since factors such as dense ground cover may suppress seedling emergence regardless of the amount of seed predation (Harper 1977), additional studies are needed to clarify the effect of seed predation on seedling emergence. Therefore, we examined the effects of both seed predation and ground cover (i.e., plant biomass and litter) on seedling emergence of some old-field forbs.
A. Extracted from: Sakoda, M., Hasegawa, K. and Ishizuka, K. 1992. Mode of action of natural growth inhibitors in radish hypocotyl elongation -- influence of raphanusanins on auxin-mediated microtubule orientation. Physiol. Plant. 84:509-513.
Seeds of Raphanus sativus L. var. hortensis f. shogoin were sown and germinated in petri dishes on 4 layers of paper-towel (Kimberly-Clark Corp.) moistened with distilled water. After 3 days in darkness at 25oC, 4-mm hypocotyl segments were excised below the hook of the 3 cm long etiolated seedlings. After subapical segments were held for 1 h in darkness at 25oC in distilled water, they were transferred to 1 mM IAA solution or mixed media containing 1 mM IAA and raphanusanin B ( 1 or 3 mM). In other experiments, segments were preincubated for 1 h in small petri dishes containing 1 mM IAA solution, and then raphanusanin B was added to the medium (final concentrations 1 or 3 mM). Segment lengths were measured using a microscope with microgauge. All manipulations were carried out under dim green light (3mW m-2).
[The authors then explained visualization of microtubules by immunofluorescence]
B. Excerpted from: Kanbe, T., Kobayashi, I and Tanaka, K. !992. Dynamics of cytoplasmic organelles in the cell cycle of the fission yeast Schizosaccharomyces pombe: Three-dimensional reconstruction from serial sections. J. Cell Sci.,94: 647-656.
Schizosaccharomyces pombe h90, the homothallic, readily sporing haploid strain, was used. The strain was maintained on malt extract-yeast extract (MY) agar as described by Tanaka and Kanbe (1986). Cells were cultured on a MY slant at 30oC for 48 h, transferred to MY broth and cultures at 30oC overnight. Cells at the exponential phase were spread on a MY plate and further incubated at 30oC for 4 to 6 h before harvesting for microscopy.
Cells were fixed with a solution of 3% paraformaldehyde in a 50mM-phosphate buffer containing 1mM-MgCl2 (pH 6.8) at room temperature for 2 h. After washing with the buffer, cells were treated with Novozyme 234 (Novo Industri A/S, Bagsvaerd, Denmark) for 60 min at 30oC with reciprocal shaking to remove the cell wall. For the staining of F-actin, cells were washed and suspended in Rh-ph solution (Molecular Probes, Inc., Eugene, OR, USA) diluted 20 times in 50 mM-phosphate-buffered saline containing 1mM-MgCl2 (PBS, pH 7.3) at room temperature for 2 h. Nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI) in NS buffer described by Suzuki et al. (1982). Preparations were examined with an Olympus BHS-RFK epifluorescence microscope using a U-G dichroic mirror with excitation filter BP490 for Rh-ph staining and UG1 for DAPI, and were photographed on Kodak Tmax400 film.
[This section continued to describe preparation for electron microscopy and the three-dimensional reconstruction of serial sections.]
A. Excerpted from: Takahashi, H., Scott, T.K. and Suge, H. 1992. Stimulation of root elongation and curvature by calcium. Plant Physiol. 98:246-252.
As shown in Table 1, the growth of roots treated with 10 mM Ca2+ was approximately 30% greater than the controls for a 3.5 h period following Ca2+ application to Alaska pea roots and approximately 80% greater than control for 12 h following the treatment in ageotropum pea. However, the growth of Alaska pea roots did not differ from that of control roots when measured 12 h after Ca2+ treatment. Roots of Silver Queen corn also showed an increase of approximately 70% in growth 3 h following application of 20 mM Ca2+ (Table 1). Such symmetrical treatment of root caps with Ca2+ did not cause curvature of the roots.
[The results section continued for several more paragraphs.]
B. Excerpted from: Sato, S. and Dickinson, H.G. 1991. The RNA content of the nucleolus and nucleolus-like inclusions in the anther of Lilium estimated by an improved RNase-gold labelling method. Jour. Cell Sci. 94:675-683.
Gold particles were predominant over the nuclear nucleolus-like bodies (NLBs) (Fig. 9). Although the distribution histogram of gold particles over the nuclear NLBs showed that labelling varied from 40 to 130 particles mm-2, most of that fell in the range of 80 - 90 particles mm-2 (Fig. 4). The quantitative estimation of labelling, which represented the average number of gold particles per mm2, indicated the labelling over the nuclear NLBs to be twice as strong as that over the loosened chromatin, and four times as strong as that over the condensed chromatin (Table 2).
[The results section continued for several more paragraphs.]
A. Excerpted from: Takahashi, H., Scott, T.K. and Suge, H. 1992. Stimulation of root elongation and curvature by calcium. Plant Physiol. 98:246-252.
The effect of Ca2+ on root elongation has been reported to be both stimulatory and inhibitory (Burstrom 1969, Evans et al. 1990, Hasenstein and Evans 1986). In those initial studies , however, the whole root was treated with Ca2+. Because the site of action for Ca2+ in gravitropism is considered to be the root cap rather than the zone of elongation, we focused on the role of the Ca2+/cap interaction in root growth as well as in gravitropic responses. We found that Ca2+ at 10 or 20 mM applied to the cap end of pea and corn roots mediated elongation growth of roots for at least 3 to 4 h following treatment. Unilateral application of 1 to 20 mM Ca2+ to the root cap always induced unequivocal curvature of roots away from the Ca2+ source in Alaska pea and to a greater extent in the roots of the agravitropic mutant, ageotropum (Figs. 1 and 2). Roots of Merit and Silver Queen corn also always curved away from Ca2+ applied to the cap, although a somewhat higher concentration was required for the response than in the pea roots. [Several sentences were omitted here.] These results show a strong correlation between an increase of Ca2+ levels in the root cap and stimulation of root elongation. The results are in contrast to the previously proposed model that an increased level of Ca2+ in the root cap mediated inhibition of root growth (Hasenstein et al. 1988).
[The discussion continued for several more paragraphs.]
A. Excerpted from: Noguchi, H. and Hasegawa, K. 1987. Phototropism in hypocotyls of radish. III. Influence of unilateral or bilateral illumination of various light intensities on phototropism and distribution of cis- and trans-raphanusanins and raphanusamide. Plant Physiol. 83: 672-675.
The present study demonstrates that phototropism in radish hypocotyls is caused by a gradient of growth inhibition which depends on the light intensity through the amounts of growth inhibitor, and thus strongly supports the Blaauw (Blaauw 1915) hypothesis, explaining phototropism as an effect of local growth inhibition by light.
B. Excerpted from: Nick, P., Bergfeld, R., Schäfer, E. and Schopfer, P. 1990. Unilateral reorientation of microtubules at the outer epidermal wall during photo- and gravitropic curvature of maize coleoptiles and sunflower hypocotyls. Planta 181: 162-168.
The striking agreement between changes in microtubule orientation observed at the outer epidermal wall during tropic bending and during induction or straight growth by external auxin strongly indicates that auxin is, in fact, functionally involved in mediating asymmetric growth leading to organ curvature.
There is no evidence that short-term growth of epidermal cells is controlled through the orientation of microfibrils. Also the data do not prove a causal relationship between auxin action on microtubule orientation and tropic curvature. However, our results do show that microtubule reorientation is a specific auxin-mediated response which can be used as a diagnostic test for an asymmetric distribution of the hormone, correlated with asymmetric growth.
New research into the spreading of infections reveals need for greater collaboration between biology and physics
Model of a social network. There are 150 individuals (the dots), whose social connections are marked by the lines between them. There are three categories: 1. Close contacts, e.g. household (yellow lines), 2. Regular contacts, e.g. work and adult friends (red lines) and 3. School contacts for children and children’s friends (orange lines). The color of the dots marks the age – darker = older. The most important knowledge derived from the research is that un-repeated contacts, e.g. from public transportation, represents a large risk of contamination in super spreader diseases like Covid19. This is why the lockdown tool, which has been widely applied in battling the pandemic, has been extraordinarily efficient. Credit: Niels Bohr Institute
Researchers at the Niels Bohr Institute, University of Copenhagen, together with epidemiologist Lone Simonsen from Roskilde University form part of the panel advising the Danish government on how to tackle the different infection-spreading situations we have all seen unfold over the past year. Researchers have modeled the spread of infections under a variety of scenarios, and the Coronavirus has proven to not follow the older models of disease spreading.
An increasingly varied picture of its behavior and thus its impact on society has emerged. In several scientific articles, researchers have described the knowledge accrued to date, most recently around the concept of "super-spreaders." It turns out that only approximately 10% of those infected account for roughly 80% of the spread of the infection. The results have been published in the scientific journal Proceedings of the National Academy of Sciences, PNAS.
Where does our knowledge of infections spreading stem from?
The data researchers use to" feed" and develop computer models comes from a wide range of different sources. The Danish municipalities have kept inventories of the spread of the infection, and this data has the advantage that it stems from units that are not overly large. There is a high degree of detail and this means that one can trace local development more clearly and thus construct parameters for super-spreading, which Postdoc Julius Kirkegaard has contributed to. Contact tracing is another source of information. In that case, the focus is on localizing and limiting the individual's transmission of the virus. The third source is slightly more complicated as it seeks to follow the chain of infections via the gene sequence of the virus.
Who are the super-spreaders?
Regardless of which source researchers examine, the results deliver roughly the same: 10% of all those infected account for as much as 80% of the spread of the infection. It is therefore crucial, in relation to the spread of the virus to locate the so-called super-spreaders and uncover how super-spreading occurs. Researchers stress that, at the moment, we are not quite sure what constitutes a person as a super-spreader. It may purely be down to personal, physiological characteristics. In addition, there are varying degrees of super-spreading in the population, so it is not necessarily just one or the other. Some people simply spread the virus more than others and the variation from persons with almost no transmission to super-spreaders is great.
How do researchers model a population of just under 6 million individuals?
Three basic categories are considered important when modeling the population's behavior, when calculating a scenario for the spread of infection: 1. The family context, 2. Work context and 3. The random contexts people find themselves in—in other words, people in proximity on public transport, at leisure activities etc. The time factor in all three is crucial, as it takes time to infect other people. In terms of time, these three categories are somewhat identical when it comes to common diseases, but not a super-spreader coronavirus variant.
But this is where the individual characteristics of the virus come into play: super-spreaders are quite different when handled in a computer model. Methods known from physics become important here, as it is necessary to model individuals and their contacts. Researchers have set up computer models both for scenarios with and without super-spreaders, and it transpires that shutting down workspaces as well as sporting events, and public transport has the same effect when the model does not take super-spreaders into account. But when we include super-spreaders, there is a pronounced difference, and the shutdown of public events has a much greater effect.
Disease modeling faces new challenges and strong interdisciplinary collaboration
Diseases can behave very differently and it therefore incredibly important to be both ready and capable of rapid change in relation to the development of new models that reflect the characteristics of different diseases as accurately as possible, if we hope to contain them. Professor Kim Sneppen explains: "The biological variation of different viruses is enormous. SARS-CoV-2 contains a special feature in that it is at its most contagious just before one develops symptoms. This is the exact opposite of an earlier disease that threatened to become a pandemic, namely SARS, which is mostly contagious after one displays symptoms. Viruses are extremely advanced machines that each find specific weak points to exploit. A new field of research is rapidly developing, which examines how viruses attack the cells in our body. COVID-19 has proven to lead to very different sickness progressions for different patients. In that senses, it behaves chaotically, as we say in physics."
Ph.D. student Bjarke Frost Nielsen and Professor Kim Sneppen see a large open field of research within the collaboration between physics and biology. Gathering as much possible information about different viruses is crucial thus enabling physists to deploy this knowledge in mapping scenarios to respond to them.
The potential for research into the spread of infections is great
Bjarke Frost Nielsen says: "We need to create a toolbox that contains a wide variation in the way we tackle the spread of transmission, in our computer programs. This is the immediate perspective we can see in front of us, at the moment. Mathematical disease modeling has been around for almost 100 years, but unfortunately not a lot of headway has been made over that period. To put it bluntly, the same equations from the 1930's are still in use today. In relation to some diseases, they can be correct, but in relation to others they can be way off. This is where, as physicists, we have a completely different approach. There are numerous parameters, i.e., social dynamics and much more varied interactions between individuals that we can build our scenarios upon. This is badly needed, when we see the enormous variations in the different diseases."
Kim Sneppen et al, Overdispersion in COVID-19 increases the effectiveness of limiting nonrepetitive contacts for transmission control, Proceedings of the National Academy of Sciences (2021). DOI: 10.1073/pnas.2016623118
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Scientific Frontiers in Developmental Toxicology and Risk Assessment (2000)
The absence of an incisive understanding of the action of toxicants on development has been in large part attributable to the absence of understanding of development itself. Until a few years ago, there was no understanding of a &ldquodevelopmental mechanism&rdquo at the molecular level although there were explanations at the cellular and tissue levels, such as &ldquogastrulation is the mechanism by which the organization of the egg is transformed into the organization of the embryo.&rdquo Recent advances in developmental biology have been substantial enough for scientists to be confident for the first time that some aspects of development in some organisms are understood at the molecular level. Protein components are identified, their functions in developmental processes are known, and the time and place in the embryo of expression of the genes encoding them are known. This knowledge greatly benefits elucidating the mechanisms of developmental toxicity.
In this chapter, the committee, in response to its charge, evaluates the state of the science for elucidating mechanisms of developmental toxicity and presents insights of developmental biology. It will show the promise of the subject in the next decade for understanding the action of developmental toxicants.
A BRIEF HISTORY OF DEVELOPMENTAL BIOLOGY
Observations of embryos and embryonic stages were made and recorded in antiquity (e.g., Aristotle, fourth century BC) and with increasing attention in recent centuries (e.g., Malphigi in the 1600s, Wolff in the 1700s, and von Baer in the early 1800s). However, it was only in the late nineteenth century that scientists pursued a detailed description of the embryonic stages of a variety of verte-
brates and invertebrates, aided by the then-recent improvements in light microscopy and in staining methods and stimulated by Darwin&rsquos proposals that the study of ontogeny (i.e., the animal&rsquos embryonic development) holds clues to phylogeny (i.e., its evolutionary origin). Among the highlights during the period of 1880-1940 were the detailed anatomical descriptions of developmental stages of embryos, including the first atlas of human embryos, reconstructed from microscopic sections, published by W. His, Sr., in 1880-1885. In vertebrate embryology, these descriptions revealed the organogenesis of the heart, kidney, limbs, central nervous system (CNS), and eyes. Developmental-fate mapping studies revealed the embryonic sites of the origin of cells of the organs and the rearrangements of groups of cells in morphogenesis. The stages of development were found to include, in reverse order, cytodifferentiation, organogenesis, morphogenesis (gastrulation and neurulation), rapid cleavage, fertilization, and gametogenesis. By the 1940s, anatomical descriptions of the embryos of related animals were integrated into coherent evolutionary schemes, taught in comparative embryology classes, revealing, for example, the modification of the gill slits of jawless fish to the jaw of jawed fish and further modification to the middle ear of mammals. Also, by this time, Haeckel&rsquos oversimplified scheme had been abandoned, namely, that ontogeny merely recapitulates phylogeny.
Experimental embryology also began in the late 1800s. In experimental studies, which mostly involved techniques of cell and tissue transplantation and removal, the central role of cytoplasmic localizations and cell-lineage-restricted developmental fates was recognized in the development of certain invertebrates by the early 1900s. In vertebrate development, the importance of inductions (also called tissue interactions) was recognized in the 1920s, following the stunning organizer transplantation experiments by Spemann and Mangold (1924) on newt embryos. By the 1950s, inductions had been found in every stage and place in the vertebrate embryo, for example, in all the kinds of organogenesis. Vertebrate development, including that of mammals, had become comprehensible as a branching succession of inductive interactions among neighboring members of an increasingly large number of different cell groups of the embryo.
Developmental mechanisms, as understood even in the 1970s, were descriptions of the movements and interactions of cells or groups of cells. They were cellular- or tissue-level mechanisms. The all-important &ldquoinducers&rdquo were materials of unknown composition released by one cell group and received by another group. Consequently, the recipient cells took a path of development different from the one that would have been taken if they were unexposed. The progression or momentum of development also was recognized: that the individual events of interactions and responses are time-critical, and that certain subsequent aspects of development never occur if one event is prevented.
Molecular mechanisms, however, were not understood at that time. Embryologists encountered the limits of the field in the 1940-1970 period, as they tried to discover the chemical nature of inducers and the responses of cells to them.
The basic information and methods of biochemistry, molecular biology, cell biology, and genetics were not yet available to analyze cell-cell signaling and transcriptional regulation in embryos. In light of discouraging results, some embryologists considered that the organizer concept was faulty and that inducers were an experimental artifact (see later discussion for recent successes in understanding inductions). Although Morgan and other early geneticists had proposed that inducers and cytoplasmic localizations elicit specific gene expression and that development was in large part a problem of ever-changing patterns of gene expression (Morgan 1934), the means were not at hand to pursue those insights. Roux, Spemann, and Harrison had outlined plausible lines of inquiry into determination and morphogenesis in the early part of the twentieth century however, the means were also not available to pursue those questions at that time.
To many scientists in the 1940-1970 period, the study of development seemed messy and intractable. Researchers turned to more informative subjects such as the new molecular genetics of bacteria and phages (viruses that infect bacteria). From those inquiries came new insights in the 1950-1965 period on the nature of the gene and the code and the processes of replication, transcription, translation, enzyme induction, and enzyme repression. For example, it was only in 1961 that Monod and Jacob described gene regulation in bacteria in terms of promoters, operators, and repressor proteins (Monod and Jacob 1961). Those authors immediately saw the relevance to animal development. All of their insights made possible the invention of techniques for gene isolation and amplification, for in vitro expression of genes, for genome analysis, and, thereafter, for the new developmental biology.
With so little molecular information about developmental processes, there was scarcely any understanding of the action of developmental toxicants. For example, Wilson (1973) in his book Environment and Birth Defects could only raise the following possibilities for connections between inductions and developmental defects:
It has long been accepted that cell interactions (induction) are an important part of normal embryogenesis, despite the fact that specific &ldquoinducer substances&rdquo have not been identified. [Failures] of normal interactions which may lead to deviations in development include, for example, lack of usual contact or proximity, as of optic vesicle with presumptive lens ectoderm or the incompetence of target tissue to be activated in spite of its usual relationship with activator tissue, as in certain mutant limb defects or the inappropriate timing of the interrelation, even though all parts are potentially competent. That the nature of cell-to-cell contacts and the manner of their adhesion are important determinants in both normal and abnormal development has been demonstrated&hellip. Insufficient or inappropriate cellular interactions usually result in arrested or deviant development in the tissue ordinarily induced or activated by the interaction.
This committee will later argue that Wilson&rsquos insight was well directed and is now ready to be pursued.
ADVANCES IN DEVELOPMENTAL BIOLOGY
In the past 15 years, developmental biology has advanced remarkably, perhaps as at no other time in the field&rsquos history. It is now known that the trillions of cells of a large animal, such as a mammal, have the same genotype, which is the same as that of the single-celled zygote (the fertilized egg) from which the animal develops. That is to say, the genetic content of somatic cells does not change during the development of most animals. The recent clonings of Dolly the lamb (Wilmut et al. 1997), the Cumulina mouse family (Wakayama et al. 1998), and a nonhuman primate (Chan et al. 2000) reaffirm the fact that a specialized cell, such as a mammary or cumulus cell, carries the genes for all other kinds of cells of the animal. The scientific advances that led to these clonings were built on earlier nuclear transplantation successes in frogs, first by Briggs and King (1952), but particularly by Gurdon (1960), which had led to similar conclusions for a nonmammalian vertebrate. Despite the same genes, the cells within the individual organism differ greatly in their appearance and functions, meaning that they have the same genotype and different phenotypes. The cell types differ greatly in the ribonucleic acids (RNAs) and proteins contained within them. They differ in which subset of genes they express from their total genomic repertoire. At least 300 cell types are recognized in humans (e.g., red blood cells, Purkinje nerve cells, and smooth or striated muscle cells). The number of cell subtypes is much larger, perhaps numbering tens of thousands, when further differences are taken into account related to the cell&rsquos stage of development and location in the body, as has been discovered in recent years. Development can be viewed as evolution&rsquos crowning example of complex gene regulation. From the single genome, thousands of different gene combinations must be expressed at specific times and places in the developing organism, and from the developing egg the information for the selective use of combinations must be generated.
A major factor in this regulation is the transfer of chemical information (i.e., signals) between cells during development. From recent research, which has built on earlier findings, the following is now realized:
Embryonic cells of arthropods and nematodes make many of their developmental decisions based on which chemical signals they receive from other cells just as vertebrate embryonic cells do. Later the embryonic cells of all these organisms will make further decisions based on other signals. The cycles of signaling and responding are repeated over and over as development progresses. With that in mind and the fact that one genotype supports hundreds or thousands of cellular phenotypes, development can be said to rely on &ldquogenotype-environment interactions,&rdquo where the local environment of each cell is generated by neighboring groups of cells. The genotype and cell&rsquos previous developmental decisions determine its options for responses to the signals currently present (Wolpert 1969).
The signaling pathways involved in this information transfer are known to be of 17 types (a few more may remain undiscovered). They are used repeatedly
at different times and places in the embryo, from the earliest stages through organogenesis and cytodifferentiation, and even in the various proliferating and renewing tissues of the juvenile and adult (see Appendix C).
The signaling pathways are highly conserved across a wide range of phyla of animals (from chordates to arthropods to roundworms), presumably because they were present and already functional in the pre-Cambrian common ancestor of those animals.
Many of the kinds of cell responses to signals also are conserved (e.g., responses of selective gene expression, secretion, cell proliferation, or cell migration). The response of developing cells to signals involves activation or repression of the expression of specific genes by transcription factors contained within genetic regulatory circuits. Signaling pathways frequently affect the activity of those factors. Many of the transcription factors and circuits are conserved across a wide range of phyla of animals.
Thus, an effective and general approach to the experimental analysis of developmental processes at all stages has been to inquire about the signaling pathways and transcriptional regulatory circuits that operate in the particular instance of development under study. Different organisms, which differ in aspects of their development, nonetheless use the same conserved signaling pathways and regulatory circuits, but in different combinations, times, and places, and have different genes as the targets of their transcriptional regulatory circuits. Processes of development, which seemed to confront scientists with infinite complexity and variety just a few years ago, now seem interpretable as composites of a small number of conserved elemental processes, namely, those of intercellular signaling, intracellular regulatory circuits, and a limited variety of targeted responses. These conclusions, which were reached by the analysis of development in animals as remote as mice, flies, and nematodes, give great validity to the use of model organisms in studying mammalian development, including that of humans, and in the future analysis of the action of developmental toxicants and in their detection.
Although the signal-response pathways are highly conserved, evolution has produced an increasing complexity of the &ldquocommunity&rdquo of pathways in vertebrates. This complexity is evident both in the increased number of closely related pathway components (diversifed protein family members) and in the increased possibilities for cross-talk among pathways. The redundant function of closely related components was made evident by existence of numerous targeted gene-knockout mutations in the mouse that produced little or no identifiable pheno-types&mdashthat is, the mice are normal or nearly normal under laboratory conditions (see Table 6-5, later in this chapter). It must be emphasized, however, that functional redundancy provides two advantages. It protects the organism by ensuring that a fundamental process can proceed even in the absence or reduced presence of a critical gene activity. On an evolutionary scale, the multiplicity of overlap-
ping functions provides a basis for generating diversity without losing essential functionality.
The Drosophila Breakthrough
The recent molecular understanding of developmental processes and components was gained from the experimental analysis of a few model organisms such as Drosophila melanogaster (the fruit fly), Caenorhabditis elegans (a free-living nematode), Danio rerio (the zebrafish), Xenopus laevis (a frog), the chick, and the mouse (see Chapter 7 for proposals about their use in the assessment of developmental toxicities). D. melanogaster and C. elegans were chosen by researchers for their amenability to genetic analysis, afforded by their small size (hence, large populations) and short life cycle (hence, many generations). Nüsslein-Volhard and Wieschaus (1980) began a systematic search for developmental mutants of Drosophila in the mid-1970s. They submitted adults to high-frequency chemical mutagenesis and then inspected large populations of offspring for mutant individuals with strong and early developmental defects (before hatching) at discrete locations and discrete stages in the embryo. They discarded mutants with weak or pleiotropic effects as ones too difficult to analyze at their start. They examined mutagenized flies until the same kinds of mutants began to appear repeatedly in their collections. The recurrence was evidence that they had obtained all the different kinds of zygotic mutants (those affected in genes transcribed after fertilization) that mutagenized flies could yield under the conditions of inspection. This procedure is called &ldquosaturation mutagenesis,&rdquo in which all the susceptible genes whose encoded products are important in development are thought to be revealed. Several laboratories, including those of Nüsslein-Volhard and Wieschaus, were also collecting maternal-effect mutants (those affected in genes transcribed in female germ cells before fertilization) and pursued this search to saturation.
The Drosophila mutants were categorized by phenotype and complementation behavior (putting two mutations together in a heterozygote to see whether they are alike or different) to establish the number of different genes whose mutations give the same phenotypic defect of development. Their categories included those embryos failing to develop the anterior or posterior end, odd or even segments, dorsal or ventral parts, mesoderm, endoderm, or nervous system. Further mutant combinations were made to establish epistasis (the interaction of different gene products, reflected in the dominance of one mutant defect over another) and to deduce plausible developmental pathways in which the actions of the encoded gene products could be related and ordered. By the late 1980s, a solid base of observations of Drosophila mutant phenotypes and gene locations had been built, and ordered pathways of function based on the mutant interactions had been proposed. This information served as the foundation for future molecular genetic analysis. The research was the first systematic and exhaustive approach to under-
standing an organism&rsquos development and to identifying components of developmental processes.
Synergy with Research Advances in Other Areas
Meanwhile, other researchers worldwide made advances in biochemistry, molecular biology, cell biology, and genetics. They learned an enormous amount about the function of proteins in replication, transcription, translation, secretion, uptake, membrane trafficking, cell motility, cell division, the cell cycle, cell adhesion, and apoptosis (programmed cell death), to mention but a few of the cellular processes. Researchers improved the methods to isolate genes, sequence them, manipulate sequences, make transcripts in vitro, detect messenger (m)RNAs in cells by in situ hybridization, translate RNAs to proteins in vitro, and make antibodies to proteins. In situ hybridization, which graphically revealed the time and place of expression of specific genes in the embryo, was to prove particularly important for connecting the new molecular analysis to the older developmental anatomy. Much of the work was initially done with single-celled organisms: bacteria, yeast, or animal cells in culture. Some insights and techniques came from the study of cancer cells in the search for oncogenes.
In the course of that work, many of the processes, protein functions, and protein sequences were found to be strongly conserved among organisms as diverse as yeast and humans or even bacteria and humans. Various proteins of different organisms, and also within the same organism, shared &ldquosequence motifs&rdquo by which the protein could be recognized as a member of a protein family with a particular function and descended from a common sequence ancestor. Newly discovered proteins could be assigned a function from just their possession of a particular motif. As more motifs were found, it will be easier to categorize newly discovered proteins. For example, receptor tyrosine kinases were recognizable by their transmembrane hydrophobic motifs and adenosine triphosphate (ATP)-binding domains. G-protein-linked receptors could be distinguished by a seven-pass (serpentine) transmembrane motif. Transcription factors could be recognized by the sequence motifs of their deoxyribnucleic acid (DNA) binding domains (e.g., zinc finger, basic helix-loop-helix, homeodomain, or leucine zipper domains). Of the recently sequenced genomes of yeast and C. elegans, for example, about 40% of the open reading frames (ORFs) are recognizable by known motifs (Chervitz et al. 1998). Function can be assigned, at least preliminarily, to the products of those genes. Plans are afoot to define the function of the missing ORFs of yeast and make the functions of all proteins assignable from sequence. At the same time, there are plans to identify a large number of protein-binding sequences in the regulatory regions of genes to be able to predict better the conditions of expression of genes. These plans are among the aims of &ldquofunctional genomics,&rdquo as described in Chapter 5. All the information on sequences, motifs, and function is stored in databases available
to researchers worldwide (e.g., the Basic Local Alignment Search Tool (BLAST) <http://www.ncbi.nlm.nih.gov/BLAST/>).
Drosophila Development at the Molecular Genetic Level
By the time the Drosophila mutants were characterized in the mid-1980s, techniques were well-suited for molecular genetic analysis of affected genes and gene products. This part of the work moved quickly, thanks to gene-cloning techniques, background information about gene sequence motifs and protein function, and databases available to researchers worldwide. The successful isolation of a gene responsible for a developmental phenotype (when the gene was mutated) could be validated by the rescue of the mutant phenotype by transformation with the wild-type gene (usually as DNA included in a P-element transposon). In situ hybridization, coupled with color stains, readily revealed the normal time and place of expression of the specific genes whose mutations had been isolated. Regarding the function of these developmental genes, many were found to encode proteins with familiar motifs, such as those for receptor tyrosine kinases or various transcription factors. In fact, a surprisingly large number turned out to be transcriptional regulators. Function could be rapidly concluded from sequence data. Other Drosophila genes encoded proteins whose specific functions were unknown, yet they were recognizable generally as secreted proteins by their signal sequences or as new transcription factors by the fact they accumulated in nuclei and could bind to DNA. In the course of this analysis, new intercellular signaling pathways were discovered, such as those involving the Decapentaplegic (DPP), Hedgehog (HH), Wingless (WG), and Notch/Delta ligands. (The whimsical names are those given by researchers to mutants based on the phenotypes.)
Hundreds of laboratories worldwide joined the work on Drosophila mutants, and the picture of early development took on a satisfying coherence and clarity, especially the steps of generation of segmentation and of the overall body organization in the anteroposterior and dorsoventral dimensions. These steps of early development are known collectively as &ldquoaxis specification.&rdquo The following is a brief summary of that picture to illustrate its completeness at the molecular level. The steps are stage-specific mechanisms of development. The mechanisms are now better understood in Drosophila than in any other organism. It is the kind of information scientists would like to have, but do not yet have, for mammalian development.
At the start of Drosophila development, the oocyte is provisioned with hundreds of maternal gene products that are uniformly distributed in the egg during oogenesis. Four gene products are spatially localized in the egg, however, and they provide the initial asymmetries on which the entire anteroposterior and dorsoventral organization of the embryo is built stepwise in development after fertilization. The four gene products include the following:
An mRNA located internally at the anterior end (encoding a transcription factor, named Bicoid).
An mRNA located internally at the posterior end (encoding an inhibitor of the translation of the mRNA for a transcription factor, named Nanos).
An external protein anchored to the egg shell at both ends of the egg (involved in the production of a ligand of a receptor tyrosine kinase in the egg-cell plasma membrane).
An external protein also anchored to the egg shell but at the prospective ventral side (involved in the production of a signal ligand of the Toll receptor in the egg-cell plasma membrane).
To exemplify the steps of use of those gene products, only one of the dimensions, the anteroposterior, will be described. The two mRNAs are initially at opposite ends of the egg. They are translated after fertilization, and the encoded proteins diffuse from the ends to form opposing gradients reaching to the middle of the egg. These proteins will act in concert to generate a gradient, high at the anterior end and low at the posterior end, of another transcription factor. The nuclear number increases rapidly in the uncleaved cytoplasm. The graded transcription factors, called members of the &ldquocoordinate class&rdquo or &ldquoegg-polarity class&rdquo of gene products, activate at least eight gap genes in nuclei along the egg&rsquos length at different positions, each position unique in terms of the local quantity of transcription factors of the coordinate class. (The terms &ldquocoordinate,&rdquo &ldquoegg polarity,&rdquo and &ldquogap&rdquo also derive from mutant phenotypes.) The encoded gap proteins, which are all transcription factors themselves, accumulate in a pattern of eight broad and partially overlapping stripes along the egg&rsquos length. The proliferating nuclei are not yet separated by cell membranes&mdashthat comes later. These proteins in turn activate at least eight pair-rule genes, all of which also encode transcription factors. Complex cis-regulatory regions of the various pair-rule genes define their expression responses to the spatially distributed gap proteins. The pair-rule proteins then activate at least 12 segment-polarity genes, some of which encode transcription factors and some of which encode secreted protein signals. The pair-rule and gap proteins together also activate eight homeobox (Hox) genes to be expressed in broad stripes, as discussed in the next section. Thus, the early steps of development involve cascades of transcription factors distributed in space according to the initial gradients of a few agents and to the expression rules contained in the complex cis-regulatory regions of genes for yet other transcription factors. These key steps are accomplished in the first 3 hours of development, mostly before cell membranes are formed and gastrulation begins, although the final elaboration of the segment-polarity and Hox genes occurs after cells form.
Once the segment-polarity genes and Hox genes are activated, they maintain their expression in cells by an auto-activating circuitry, in some cases by the encoded transcription factor activating expression of its own gene. The coordi-
nate, gap, and pair-rule proteins are then no longer needed. Their products disappear, and the genes are no longer expressed.
Similar conclusions apply to the development of the termini and the dorsoventral dimension, which also rely on initially asymmetric signals. The developmental mechanisms of the termini and dorsoventral dimension are of additional interest, because the signals bind to transmembrane receptors and activate signal transduction pathways, eventually leading to the activation of transcription factors and new gene expression. These inductions are the first to occur in the developing Drosophila egg. Approximately 100 genes and encoded gene products have been identified as necessary to establish the organization of the early gastrula. Hundreds more participate in the accomplishment of these events, but they are less well described at present. In most cases, these genes probably encode proteins required in numerous developmental processes and, hence, were not recovered under the conditions of the mutant inspections used here.
As shown in Figure 6-1A-D, a coherent scheme of early development was proposed and well supported by 1992, the first of such complexity and completeness at the molecular level for any organism.
FIGURE 6-1A Outline of anteroposterior development in Drosophila and the steps of regulated gene expression (Ingham 1989). Heavy dashed arrows indicate the activation of specific gene expression by transcription factors. Thin solid arrows indicate transcription and translation. Note that Hox genes are activated by both pair-rule and gap proteins, whereas segment-polarity genes are activated by pair-rule proteins alone. In the anteroposterior dimension, segments and HOX domains are formed. Further explanation is given in Figure 6-1B.
FIGURE 6-1B Anteroposterior development in Drosophila (Nüsslein-Volhard 1991). Figure 6-1B is shown diagrammatically here, for segment formation and HOX compartment formation. The coordinate proteins Bicoid, Nanos, and Cad are translated from mRNAs localized at the two poles of the egg during oogenesis. Translation generates gradients of proteins. Bicoid and Cad are transcription factors, whereas Nanos protein inhibits the translation of another translation factor (Hunchback) in the posterior half of the egg. The graded transcription factors activate eight gap genes, and different factor concentrations activate different gap genes. The gap proteins are also transcription factors. Each diffuses locally and inhibits other gap genes, setting up eight partially overlapping stripes of gap protein along the egg&rsquos length. The gap proteins activate eight pair-rule genes, each of which has a complex cis-regulatory region and is activated by seven combinations of gap proteins, each making seven evenly spaced stripes of protein. Thus, there are 8 × 7 or 56 stripes of pair rules along the egg&rsquos length, arranged in 7-fold repeats. The pair-rule proteins are all transcription factors. These activate eight segment-polarity genes, each of which has a complex cis-regulatory region activated by at least two combinations of pair-rule proteins, to give 14 stripes of expression each. Thus, there are 14 × 8 or 104 stripes of segment-polarity proteins. The 14-fold repeat is the basis for 14 segments of the posterior head, thorax, and abdomen. The pair-rule and gap proteins together activate Hox genes in eight domains in the posterior head, thorax, and abdomen. Cell outlines are not shown, but cells are present in the two lowest panels.
FIGURE 6-1C Dorsoventral development in Drosophila (Nüsslein-Volhard 1991). The egg shell contains Pipe protein on the future ventral side, deposited there during oogenesis. After fertilization, the egg secretes several proteins into the space between the egg shell and plasma membrane. Pipe activates one of the proteins, which then sets off others in a protease cascade, the last member of which cleaves the Spätzle protein, releasing a ligand that binds to the Toll transmembrane receptor, which is uniformly distributed over the egg surface but ligand-activated only on one side. The activated receptor, via several intracellular steps, activates the Dorsal protein, a transcription factor, which enters local nuclei and activates two genes, Twist and Snail, which also encode transcription factors. Those activate other genes for gastrulation and for mesoderm formation on the ventral side. Thus, the Pipe protein is involved in a kind of mesoderm induction. Active Dorsal protein also represses the Zen and Dpp genes on the ventral side. On the dorsal side, Dorsal protein remains inactive and the Zen and Dpp genes are expressed. Laterally, there is enough active Dorsal protein to repress Zen and Dpp but not enough to activate Twist and Snail. Here, the Sog gene is permissively expressed and not repressed, preparatory to neurogenic ectoderm formation. Thus, the dorsoventral dimension of the egg is divided into three domains of gene expression. Later, the Sog protein is secreted and diffuses to the Zen, Dpp region, inhibiting Dpp signaling and allowing the division of that region into two subregions the prospective amnioserosa and prospective dorsal ectoderm.
FIGURE 6-1D The development of termini in Drosophila (Nüsslein-Volhard 1991). The Torso-like protein is present in the egg shell at the two ends of the egg, deposited there during oogenesis. After fertilization, the egg secretes several proteins into the space between the plasma membrane and egg shell. The proteins include proteases that are locally activated at the end by the Torso-like protein and release a ligand that binds locally to the transmembrane Torso receptor, a member of the RTK signal transduction family. The activated receptor locally activates Raf and MAPK, which phosphorylate a transcription factor locally, inhibiting its repression of genes and allowing local expression of the Tailless (Tll) and Huckebein (Hkb) genes involved in formation of the endoderm, terminal ectoderm, and gut involution during gastrulation. Thus, the Torso-like protein is involved in endoderm induction.
Hox Genes and the Drosophila Connection to Vertebrate Development
Even though researchers in other areas widely appreciated the breakthroughs in Drosophila development, they questioned the relevance of the information to vertebrate development. Vertebrates, as chordates, were thought to have branched from arthropods long ago and last shared a very simple common ancestor in the pre-Cambrian era (about 540 million years ago). The two groups were thought to have evolved their segmentation and heads independently. One of the first significant similarities between vertebrate and fly development came from work on homeotic genes, now called Hox genes. As mentioned before, the Hox genes are expressed in eight broad bands or spatial compartments in the anteroposterior dimension of the body shortly after gastrulation but prior to organogenesis and cytodifferentiation. Their encoded products make each spatial compartment different from the others.
The study of the eight Hox genes of Drosophila was primarily pioneered by E. Lewis from 1940 to 1970. For his work in that area, he shared the Nobel Prize
with Nüsslein-Volhard and Wieschaus in 1995. Lewis selected Drosophila mutants that exhibited mislocated body parts (e.g., wings in place of halteres (balancing organs) and legs in place of antennae). The term &ldquohomeotic&rdquo connotes such mislocation without distortion. In the homeotic mutant, the anteroposterior dimension of the animal has fewer anatomical differences along its length. For example, the Ubx mutant has an extra mesothorax located at the normal metathorax position but lacks a metathorax. It has four wings but no halteres, whereas normal Drosophila have two wings and two halteres. When the first two Hox genes (Ubx and Antp) were isolated, their sequences were compared (McGinnis et al. 1984a,b Weiner et al. 1984), and a shared 60-base sequence was found, the homeobox. The sequence is the same in both genes except for a few bases. That sequence encodes the DNA-binding motif of the encoded proteins, which are members of a large and ancient family of transcription factors. The other six Hox genes were soon isolated from Drosophila, and those too had closely related homeobox sequences. Then the eight genes were shown to exist in a contiguous cluster (actually two subclusters in D. melanogaster but one in another arthropod, Tribolium), probably all tandemly duplicated and diverged from a few founder sequences in an ancestor of arthropods. Furthermore, the members are expressed in stripes in the anteroposterior dimension of the body, in an order identical to their gene order on the chromosome (a correspondence referred to as &ldquocolinearity&rdquo of gene order and expression).
In the mid-1980s frogs and mice were found to contain similar sequences, also arranged in contiguous gene clusters. Interestingly, their expression in mice showed the same anteroposterior colinearity as that in Drosophila. As an evolutionary explanation, the common ancestor of arthropods and chordates must have had a complex Hox cluster already functioning in its development. Vertebrates, however, differ from arthropods in having at least four multi-member clusters instead of one (Krumlauf 1994). A comparison of gene arrangements and domains of expression in Drosophila and mammalian (mouse) Hox clusters is shown in Figure 6-2.
Such genes are called selector genes because their encoded products, which are transcription factors, select which other genes will be expressed in that spatial compartment of the body. The thousands of target genes of a selector-gene product encode proteins involved in subsequent local development, including the many kinds of organogenesis of different parts of the body. Hox genes have a central role in development. Because of them, the coordinate, gap, and pair-rule proteins of early development do not have to directly activate those thousands of target genes in a region-specific way but activate only the Hox genes, whose encoded proteins then do the job of regulating sets of genes in their respective regions. Methods for the directed knockout of genes in mice were invented by the mid-1980s as a way to test gene function, and the Hox genes of mice were found to control aspects of local development in their compartments, especially in vertebrae, neural tube, and neural crest derivatives. Their selector role was similar to
FIGURE 6-2 This figure illustrates the striking similarities of gene organization and expression of Hox clusters in Drosophila and mammalian (mouse) embryos. At the top is a 10-hour Drosophila embryo showing expression zones of individual Hox genes in thoracic (T1-3) and abdominal (A1-9) segments and parts of the head (Lab, labrum Mx, maxillary Ma, mandible Int, intercalary segment). Note the colinearity of Hox gene expression sites along the anterior-posterior body axis to their 3&prime to 5&prime location along the chromosome. The greatly expanded vertebrate Hox gene family is shown in the middle. These genes are arranged in four clusters (labeled A, B, C, D), each on a separate chromosome. Having arisen by duplications early in chordate evolution, Hox genes in paralogous groups (e.g., A4, B4, C4, D4 shown enclosed in dashed boxes) are more closely related than are adjacent genes (e.g., B3 vs. B4 vs. B5). The four most 5&prime paralogous groups have no close equivalent in arthropods these are expressed in the tail and fins or limbs. Lines extending from each paralogous group to the schematic brain and cranial spinal cord show the rostral limits of expression of members on each group. Note, again, the colinearity between expression sites and relative chromosomal position of most Hox genes. The same is generally true for somites and, in the proximo-distal orientation, for limbs.
that in Drosophila (Behringer et al. 1993). However, many of the target genes of Hox proteins in mice and flies are clearly different.
The Hox clusters of Drosophila and chordates are under intense study. It is now known that genes of four mouse clusters are coordinated in an elaborate circuitry of auto- and cross-activation and repression, in which the genes near the 5&prime end of the DNA sequence tend to repress genes near the 3&prime end when both are initially expressed in same cell. Equivalent paralogs in different clusters tend to overlap in the target genes they activate and repress, but each has some unique targets, as shown by the phenotypes of single-Hox knockout mutants of the mouse. As a whole, the Hox genes operate as a complex genetic regulatory system rather than as independent members.
More recently, the Hox-like Ems and Otd genes have been discovered in Drosophila as expressed in the head in regions anterior to the expression compartments of the Hox genes. Homologs of these genes (called Emx and Otx) have been found expressed in the head of the frog and mouse anterior to the Hox gene domains of the posterior head, thorax, and trunk. This was a surprise, because evolutionary biologists had thought that the vertebrate head is unique to that group and has little in common with the head of a common ancestor of vertebrates and arthropods. However, even that complexity of body organization, like HOX compartments, must predate the branching of arthropods and chordates.
The Emergence of Caenorhabditis elegans
The free-living nematode Caenorhabditis elegans emerged as an important model system in the 1970s, as the result of pioneering work on its genetics by S. Brenner (1974). Chosen for its short life cycle (3 days) and general amenability for genetic analysis, small size (1-mm length), transparency, and simplicity (only 959 somatic cells), C. elegans quickly attracted a following among developmental biologists and geneticists. In particular, J. Sulston was primarily responsible for first describing the complete cell lineage from fertilization to adulthood (Sulston and Horvitz 1977 Sulston et al. 1983) and then spearheading the physical mapping and DNA sequencing of the genome. C. elegans recently became the first metazoan organism whose genome is completely sequenced (C.elegans Sequencing Consortium 1998). In the meantime, researchers from many laboratories isolated mutants and identified many important genes controlling development, the result being that C. elegans is now the most completely described and one of the best understood models for development (see Chapter 7). In some ways, the development of vertebrates is more similar to that of C. elegans than of Drosophila (e.g., having a cellular rather than a syncyctial early embryo), and in other ways less similar (e.g., having a highly invariant cell lineage and a fixed small number of cells, no Sonic Hedgehog signaling pathway, and few HOX genes). These two model animals complement each other usefully for research into fundamental mechanisms of metazoan development.
Conserved Developmental Processes
Researchers increasingly suspected similarities of development between fruit flies and mice and began to look systematically for homologs of Drosophila developmental genes in mice, frogs, and chicks. In the late 1980s, this was a new research approach. Its success has favored the impression that at a gross level, nematodes, flies, and mice are &ldquoall the same organism&rdquo and that what is learned about one will have relevance to the others. In a genetically tractable organism, such as Drosophila or C. elegans, a gene is isolated by using a screen for a particular kind of developmental failure, and then the role of its encoded product in development is efficiently deciphered in that organism. Homologs of &ldquodevelopmentally interesting&rdquo genes are then sought in vertebrates, such as mice or frogs, in which mutant searches are still daunting due to the comparatively small populations and slow development. The homolog&rsquos function is thereafter studied in the vertebrate, for which the Drosophila or C. elegans information is used as a guide. The mouse is attractive for such studies, because the homologous gene can be knocked out and the phenotype of the null mutant examined to learn about the function of the encoded product.
A surprising array of developmental components and processes is shared between Drosophila and vertebrates (i.e., between arthropods and chordates). In addition to the EMX, OTX, and HOX organization of the body plan, they share the compartments of the dorsoventral dimension (which are thought to be inverted in orientation in one group relative to the other) the presence and mode of organogenesis of limbs (appendages), eyes, heart, visceral mesoderm, and gut the steps of cytodifferentiation during neurogenesis and myogenesis and even segmentation. Although the anatomical structures themselves are very different between arthropods and chordates, a number of the underlying steps of development are the same. These are listed in more detail in Table 6-1. The last common ancestor of chordates and arthropods was, it seems, a pre-Cambrian animal of much greater complexity than previously realized. Divergent groups of metazoa (members of the animal kingdom) can be treated as &ldquothe same organism&rdquo in the experimental analysis of many fundamentals of development. From all of those similarities, the value of model systems for gaining an understanding of difficult basic problems in mammalian development, including that of humans, is undeniable. Humans, flies, and even roundworms are less different than widely thought just 10 years ago.
Signaling Pathways in Development
An important realization to come from the Drosophila research concerns the pervasive use of cell-cell signaling in most aspects of development, starting with the termini and dorsoventral dimension (see Figures 6-1A-D) and extending to organogenesis of many kinds. Inductive signaling was thought to be important in vertebrate development, as mentioned above, but insects and other invertebrates
TABLE 6-1 Similarities of Arthropods and Chordates
Organisms That Share Process
Hox gene complex: similar order of genes in the cluster and similar order of expression domains in the posterior head and trunk (thorax and abdomen)
Anterior head organization
Ems-Otd (Emx-Otx) selector genes: similar nesting expression domains in the anterior head
Sog-Dpp-Tolloid (Chordin-BMP2,4-xolloid): similar gene expression domains, similar protein interactions in the neural versus epidermal regions similar gene expression domains in the visceral mesoderm and heart. Was the chordate dorsoventral axis formed by inverting the axis of an arthropod ancestor?
Engrailed and HH-SHH expression domains are similar in posterior half of segment or somite Hairy gene expression in alternate segments or somites
Drosophila, amphioxus, and zebrafish
Appendage or limb patterning
Similar domains of WG-HH-DPP (WNT-SHH-BMP) signaling and expression of En, Ap (En, Lmx) selector genes
Drosophila, chick, and mouse
Similar domains of expression of Eyeless-Pax6 and Sine oculis-eye selector genes
Drosophila, mouse, and human
Note: Although the organisms of these two phyla seem very different (e.g.,insects and crustaceans versus fish and mammals), they share many developmental processes at the level of their use of combinations of signaling pathways and genetic regulatory circuits. In italics are various similar conserved genes used in the conserved processes. These similarities serve as evidence that the pre-Cambrian common ancestor of chordates and arthropods was already complex in its anteroposterior and dorsoventral organization and perhaps segmented. Many aspects of cytodifferentiation are also similar (e.g., the use of MyoD in muscle and Achaete-Scute in nerve cells).
had been assumed to develop as composites of independent lineages of cells (&ldquomosaic&rdquo development). This is not at all the case. Six signaling pathways are used repeatedly in early Drosophila development: the Hedgehog, Wingless-Int (Wnt), transforming growth factor &beta (TGF&beta), Notch, receptor tyrosine kinase (RTK), and cytokine receptor (cytoplasmic tyrosine kinase) pathways. Comparative studies soon showed that these pathways exist in vertebrates as well, and most also exist in nematodes (except the Hedgehog pathway). Four other conserved pathways in addition to those six are used heavily in later development, mainly in organogenesis, and seven others come into use in the physiological functioning of the organism&rsquos differentiated cell types. The number of known pathways has now reached 17. Each pathway is distinguished by its unique set of transduction protein intermediates. The 17 pathways are listed in Table 6-2. Details of the components and steps of the individual pathways are given in Appendix C.
As a generalization, most of the pathways involve transmembrane receptor proteins that bind ligands at the extracellular face, as diagramed in Figure 6-3. Ligands arrive in some cases by free diffusion after secretion from distant neighbor cells. Others diffuse only short distances or remain attached to the surface of the cell of origin, reaching only the contacting cells. Activated receptors of the recipient cell activate the first intracellular component of a signal transduction pathway, and this then activates a subsequent component, and so on. Some pathways are long, with 7-10 intermediates. Others have one or two. The nuclear hormone receptor pathway is the shortest, having only one step. In this case, hydrophobic ligands penetrate the cell membrane on their own and bind to a receptor protein, which also functions as a transcription factor. In the longer pathways, a change of activity is passed along a series of on-off switches, which constitutes an information relay pathway, or signal transduction pathway. Ultimately, in some pathways, a protein kinase is activated at the end of the series, and that enzyme phosphorylates numerous target proteins, which change their activity (activated or inhibited) because of the phosphate addition. The target proteins are components of various basic cell processes, such as transcription, the cell cycle, motility, or secretion. Hence, these processes are turned on or off, and the change of function constitutes the cell&rsquos response to a signal. In many other pathways, a specific transcription factor is activated at the end of the pathway, and this factor is a pathway component. In development, the most frequent target of signaling pathways is indeed transcription. The pathways used in early development tend to have transcription as the only target. That is, particular transcription factors are phosphorylated or proteolyzed as a signal transduction step of the pathway, changing their activity in activating or repressing particular genes.
The pathways are used repeatedly at different times and places of development in Drosophila, nematode, and vertebrates, as listed in Table 6-3. Drosophila null mutants are usually lethal if they lack a step in any of those pathways. Lethality is an indication of the essentiality of those signaling functions. However, in the mouse (and probably all vertebrates), a null mutant for a step of a
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