Good Term Paper On Christiane Nusslein-Volhard
German biologist, Christiane Nusslein-Volhard is renowned for elucidation of the genetic control of embryonic development of fruit flies. Her contribution earned her the Nobel Prize in Physiology or Medicine, together with American geneticists Eric Wieschaus and Edward B. Lewis. Throughout this paper I will detail the early career of Dr. Nusslein-Volhard and the ways in which her research helped to revolutionize the field of developmental biology as it pertains to embryonic development and gene analysis/function.
Christiane Nusslein-Volhard was born on October 20th, 1940 in Magdeburg, Germany. As she grew, she developed interest in studying plants and animals and inclination to be a biologist by the age of twelve. In 1964, she completed her undergraduate degrees in Biology, Chemistry, and Physics at Johann-Wolfgang-Goethe University in Frankfurt, Germany. She graduated with a degree in Biochemistry in 1968 and received her Doctorate of Biology/Genetics in 1973 from Eberhard-Karl University of Tubingen. In 1969, Nusslein-Volhard began her doctoral work in Heinz-Schaller’s Laboratory at the Max Planck Institute for Virus Research. She launched her thesis work on phage RNA-DNA interactions, but quickly discovered limitations with the tools that were available at the time for experimentation (Resnik 2012). During her work, she developed a technique for RNA polymerase purification. RNA polymerase is the enzyme that transcribes RNA from DNA and binds DNA at promoter regions. Promoters were characterized by her describing the molecular process behind transcription activation. She along with one another graduate student, Bertold Heyden, studied the promoter structure by identifying the RNA polymerase binding site from fd phage. As DNA sequencing was not developed at those times, the sequence was established by oligopyrimidine pattern (Nobel, 1996). After graduating, she soon became clear that her future research work will be to study the developmental genetics of Drosophila.
Working under Dr Walter Gehring
After losing interest in molecular biology she decided to apply her knowledge of genetics to cellular biology, more specifically pattern formation and regeneration in the hydra, a genus of freshwater polyps. Then in 1973, she met Walter Gehring in Freiburg, Germany. She learned about his work with experiments in bicaudal organisms and wanted to learn more about his research (Resnik 2012). Gehring was the renowned developmental biologists of those times. She was scared at first to approach him and learn more about these new techniques but eventually she got the courage to ask him more. Gehring offered her a post doctorate position in his laboratory in Basel, Switzerland in 1975. Then she applied for an EMBO fellowship in Heidelberg, Germany that supported her research. This proved as a turning point in the career of Nusslein-Volhard. Christiane began examining a cohort of flies that did not have the bicaudal gene, therefore she was able to determine all the other genes that were involved in the mutant bicaudal phenotype (Resnik 2012). She later admitted that this was by far the most challenging gene that she had researched in her career. However, she had a breakthrough discovering a second gene that seemed to influence pattern formation in the embryos of flies, a gene later called dorsal. Christiane worked with her mentor and other postdocs on her first publication in developmental biology. Her main work with bicaudal allowed her to publish a paper in 1977, “Genetic analysis of pattern-formation in the embryo of Drosophila melanogaster.” The dissertation topic focused on genetic patterns in Drosophila embryos and the various mutations that can occur throughout development (Resnik 2012).
Pivotal work on D. melanogaster developmental genes with Dr Wieschaus
In 1978, Christiane began working in the European Molecular Biology Organization (EMBO) with Eric Wieschaus. Eric had just finished his thesis at that time in Walter Gehring’s lab. His thesis was on imaginal disc cells. Overall, when she began working in this lab with Wieschaus she discovered new ways of working on recombinant DNA and molecular biology with the focus to clone developmentally interesting genes. Soon after she joined the lab, Wieschaus left for doing his postdoc in Zurich under Rolf Nothiger. She gained much knowledge on Drosophila genetics by Jeanette Holden, a renowned geneticist (Nobel, 1996). Christiane admitted that she had a difficult time at first communicating with her team. She learned to have conversations with the postdocs in English and enjoyed the Swiss language.
Thereafter Christiane worked with an insect embryologist Klaus Sander in the year 1977. There she examined the segmental patterns of Drosophila larvae. In 1978 Christiane and Eric Wieschaus got offers from European Molecular Biology Laboratory, Heidelberg at the same time and worked there for three years from 1978-1980. At this time, Christiane felt that Wieschaus was much more experienced than her and she got the offer from EMBL because of him. He was working on flies and carried out original projects on sex determination, lineage and germ line individually. His earlier knowledge on working with fruit flies gave them hands on experience that made the significant research work that they conducted on Drosophila segmentation genes to grab them the prestigious Nobel prize. They both led independent projects initially, and then the first joint project was on kruppel. They collected mutants with segment numbers on which they did the shelf screen where they discovered new phenotypes. By generating mutations in the fruit flies they addressed the importance and necessity of genes that control development. They gradually identified the genes that govern the developmental decisions in the embryonic development of fruit flies. They both evaluated the pattern of segments and pin-pointed that specific genes were involved in different processes during development based on the type of mutation that was created within the phenotype, such as fewer segments, gaps in the normal pattern and alterations in the patterns of denticles on the segments. They spent a year studying embryos of fruit flies patterns with different mutations. They used a double headed microscope to study the samples at the same time and analyzed tens of thousands of fruit flies and it was then they identified 15 different genes that control how body parts are formed. They discovered the formation of fruit flies body segments, the relationship between humans & other vertebrates, and because of this, their research helped the scientific community understand how our own bodies are formed and how birth defects can occur.
This research was finally published in October 1980 in Nature titled as "Mutations Affecting Segment Number and Polarity in Drosophila”, while Christiane and Eric Wieschaus were working at EMBL. However they did not get much support and appreciation from fellows at the institute regarding their revolutionary research conducted there. It was pivotal as it widened the scope of research in development biology and thus they were awarded the Nobel prize after fifteen years for their study. It is important to mention here that the collaboration of Christiane and Wieschaus only led to this major finding as their knowledge complemented the screening and research work conducted without which the outcome would not have been possible. They share equal potentialities in the research that was carried out.
Other scientists that received the Nobel Prize in the last century were John O’Keefe, May-Britt Moser, Edvard Moser, in 2014. Also, James E. Rothman, Randy W. Schekman and Thomas C. Sudhof also received the Nobel Prize in 2013 (Resnik 2012).
Working on Zebrafish
Before Christiane was awarded the Nobel Prize she switched her focus from Drosophila to Zebrafish due to the simplicity of the organism and the ways she could study their physiology. Her Nobel Prize award helped her with her transition to zebrafish as her research model of choice for her future work in developmental biology (Resnik 2012).
Christiane decided to leave EMBL in 1981 even though she was offered an extension to stay but she felt uncomfortable continuing to work in the lab after not getting much support with the research done by her and Eric (Resnik 2012). He also left EMBL and pursued jobs in the US based on his earlier work. In 1981, she accepted a junior position at Friedrich-Miescher- Laboratory of the Max-Planck-Society in Tubingen. Here, she continued working on screens of maternal mutants and identified several dorsal genes. In 1985, she became the Director of Developmental Biology and continued working on molecular biology analyzing the localization of bicoid RNA. It was aided by the antibody against bicoid protein that was prepared by Wolfgang Driever. Through this, the expression pattern of segmentation genes was revealed using bicoid protein gradient. The details of this research were published in 1988 which will be discussed later.
It was in 1984 when Christiane got interested in the study on Daniorerio, zebrafish and finally she began working on it in early 1990s. The zebrafish, Daniorerio, owing its name to the striking stereotypic pattern of horizontal blue and golden stripes, has emerged as the model organism for the genetic analysis of colour pattern formation in vertebrates. During the last years an increasing number of adult viable mutants with altered color patterns have been collected, and novel approaches in lineage tracing in individual fish were developed, providing the unique opportunity to access the genetic and cell biological background of the complex and protracted developmental process in this species (Nobel, 1996). Christiane described the color patterns as having a role in kin recognition and in mate selection. Color patterns are highly variable and evolved rapidly, and as direct targets of natural and sexual selection that had a tremendous impact and highly evolutionary relevance. An understanding of the mechanisms that underlie pigmentation and color formation was an important part of understanding biodiversity and the Zebrafish as a model. She discovered that working with zebrafish allowed for a rapid rate of reproduction, clear embryos, and showed close relationships with vertebrates. She studied on the migration of cells from their site of origin to their sites of destination within zebrafish embryos.
Christiane has over 20 awards for her research in developmental biology. While Christiane was at the Max Planck Institute for Developmental biology, she had many publications focusing on Zebrafish (Resnik 2012).
An overview of the paper by Christiane Nusslein-Volhard and Wolfgang Driever (1988)
Christiane Nusslein-Volhard and Wolfgang Driever in 1988 released their findings on the bicoid (bcd) gene product under the title, “A Gradient of bicoid Protein in Drosophila Embryos” in the journal Cell. They showed that the bicoid (bcd) gene product is a 55 kd protein that is translated into protein following fertilization in Drosophila (Driever and Nüsslein-Volhard, 1988). The authors here wanted to prove that the distribution of the bicoid protein occurs first at the anterior pole of the embryo and then will decrease in concentration subsequently moving towards the posterior end. It can be explained by the morphogen effects within the embryo (Driever and Nüsslein-Volhard, 1988). The importance of these experimental methods that surround the understanding of the effects and localization patterns of bcd, proved to be very important in the world of developmental biology (Driever and Nüsslein-Volhard, 1988). It was not stated previously that at what time in the development, different protein gradients are established in the embryo like whether during egg deposition, pole formation, and cellularization/gastrulation. Christiane Nusslein-Volhard and other scientists had long studied properties of bcd mRNA and its importance creating the structural pattern of the embryo by comparing mutant embryos with wild-type embryos via transplantation experiments (Driever and Nüsslein-Volhard, 1988). The role of bcd as an anterior morphogen and possible zygotic gene was also heavily discussed at this time. This paper elaborates on the timing- onset of bcd translation at various time intervals, the gradient in unfertilized eggs using immunostaining techniques and also proposes a question regarding the molecular basis of a series of discrete states of activated genes (Driever and Nüsslein-Volhard, 1988).
The bicoid (bcd) protein
The bicoid gene is a well-known maternal patterning gene that was discovered in female-sterile mutants in Drosophila. The function was deemed important in the developmental pattern of the embryo, namely establishing the larval head and thorax. In addition to this, the bcd protein was also shown to serve as the first identifiable morphogen, the concentration gradient was shown to be important in establishing positional information, specifically in the anterior half of the embryo where it will differentially activate segmentation genes, such as gap genes. This gradient activity provided the first experimental proof of the French Flag model of pattern formation proposed by Wolpert (Wolpert, 1969).
Driever and Nusslein-Volhard’s experimental work in this paper, not only addressed but demonstrated that the Bcd protein forms an exponential concentration gradient with the maximum being at the anterior pole, reaching background levels in the posterior third of embryos at the early nuclear cycle 14 (Driever and Nüsslein-Volhard, 1988). They proposed that the exponential gradient is generated from a bcd mRNA source, localized in the anterior-most portion of the embryo, by diffusion and dispersed degradation of the Bcd protein. The problem that others in the scientific community had with this experiment was that Driever and Nusslein-Volhard did not provide evidence to support the main argument of their model, which was the localization of the bcd mRNA at the anterior pole.
Proposed morphogen models
A morphogen is a diffusible chemical signal that creates a concentration gradient that further decides upon the cellular differentiation. They are released during early embryonic development and decide the fate of tissue. Since early times, people reached the idea of morphogen after a sequence of events. At first, it was characterized that a multicellular organism results from cellularization of a single fertilized cell. Then the complex organization of embryo was explored. Then Morgan in 1901 during the workings with Drosophila proposed that the regeneration phenomenon and organism polarity are influenced by gradients of “formative substance” that later came to be known as morphogens. Spemann & Mangold in 1924 suggested the action of signals i.e. morphogens that are released from the localized organizing centers and cause cell differentiation. In due times, two significant morphogenesis models were proposed, first being the reaction-diffusion model by Turner and second being the French Flag model by Wolpert. Morphogen gradients have been studied in sea urchin, hydra, fish, snails, fruit fly so far.
Turner in 1952 stated the significant reaction-diffusion model where he for the first time addressed the term morphogens. He proposed that two different interacting morphogens with somewhat different diffusing properties can establish chemical gradient via reacting and diffusing. These two substances react by auto and cross catalyzing or by inhibiting their production creating spatial patterns with variant morphogen concentrations.
Wolpert in 1969 then explored the idea of “organizing centers” and proposed the French Flag model. It states that the morphogen is secreted by the production or source cells and forms a localized gradient at the target site. Then the target genes are expressed in proportionality with the morphogen concentration generating a spatial pattern. It uses the tricolor flag of France to show that the embryonic cells create similar patterns using genetic code even when a part of embryo is removed. Later, quantitative models have also been proposed to understand the pattern formation.
Experiment # 1
The first experiment in this paper showed how the bcd gene product, a 55 kd protein, is translated soon after egg deposition. In order to identify and characterize bcd in Drosophila embryonic extracts, rabbit polyclonal antibodies were used against a LacZ-bcd fusion protein that contained the longest open reading from of the most abundant transcript, which was 378 amino acids in length (Driever and Nüsslein-Volhard, 1988). They used extracted protein from staged embryos, and then separated them by SDS-PAGE, and analyzed Western blots by probing with monoclonal anti-bcd antibodies. The proteins corresponded with a doublet of proteins that had molecular weights between 55 and 57 kd which were recognized by the polyclonal rabbit antibodies (Driever and Nüsslein-Volhard, 1988). The size of the protein corresponded very well with the actual predicted molecular mass of bcd, which was stated to be 53.9 kd (Driever and Nüsslein-Volhard, 1988). The antibodies were used to raise against bcd fusion proteins that were recognized by a 55-57 kd doublet band in Western blots of extracts of 0-4 hr old embryos. Once this was determined, they tried to detect bcd protein in hemizygous females, Figure 3. The immunologically detectable protein pattern was altered only in the 55-57 kd range, and none of the strong alleles (E1, E2, GB, 23-16, 33-5), gave rise to a protein in the bcd size range. The sensitivity of this method may preclude the detection of a severely truncated or very unstable protein with the antibody (Driever and Nüsslein-Volhard, 1988). Figure 2 shows the developmental profile of the bcd protein in Drosophila embryonic extracts. The time at which the 55 and 57 kd proteins are detectable at their highest abundance (2-4 hr after egg deposition) has been shown to correspond to the temperature sensitive period of the bcdE3 allele (Frohnhofer and Nusselein-Volhard, 1987). The immunostain detected protein bands that showed a maximum in staining intensity between 2 and 4 hours after egg deposition. One of the key findings was that the proteins were not detectable in ovaries or at later stages in development, in 9 of the 11 bcd alleles that were used.
Experiment # 2
The additional experiment that was conducted for this paper revolved around the distribution of bcd protein in the early embryo. An anti-bcd antibody was used to detect when the protein was first detectable (Figure 4A) at the anterior tip of the egg or near the site where localization of mRNA was proposed to occur. It was discovered that the shape of the gradient appeared to be constant during the syncytial blastoderm stage, with the posterior limit of detection being at about 30% of the egg length (Figures 4C and 4D) (Driever and Nüsslein-Volhard, 1988). It was noticed that the highest amount of bcd that was detectable appeared to increase until cellularization/gastrulation occurs (Figure 4E and 4F)(Driever and Nüsslein-Volhard, 1988). It is known that bcd is a protein that is present in the cytoplasm but predominantly concentrated in the nuclei. This proves to be important for DNA binding proteins and translation events. It was noted that during mitosis, the staining disappeared completely from nuclear structures and for a short time (Figure 4G), the embryos displayed protein gradients with no cellular substructures that were stained. An important consideration/theory that is proposed regarding the onset of bcd translation lies within the oogenesis(Driever and Nüsslein-Volhard, 1988). Since the bcd mRNA is already present during oogenesis (Friegerio et al., 1986; Berleth et al., 1988), this indicates that bcd mRNA translation is blocked during oogenesis. The biological significance of why translational control occurs at all is not entirely obvious in the developmental biology world (Driever and Nüsslein-Volhard, 1988). Since the bcd protein concentration increases during the early hours of embryogenesis; the final shape of the gradient may depend on the timing of bcd mRNA translation (Driever and Nüsslein-Volhard, 1988).
The shape of the bcd concentration gradient was determined by using immunostaining techniques with nitrocellulose. The intensity of the bcd protein immunostain was measured from the anterior to posterior of the embryo and is displayed in Figure 6A. The bcd protein was shown to be distributed in a sharp nonlinear concentration gradient (Driever and Nüsslein-Volhard, 1988). The result of these measurements showed that there was an exponential decay of the concentration of the protein towards the posterior. In addition, it is estimated that background levels are reached at about 30% of the egg length (Figures 6B and 6F). The shape of the bcd protein gradient in unfertilized eggs was also addressed at length and can be seen in Figure 6E, that unfertilized eggs are similar to that in fertilized eggs.
One aspect of the paper that was discussed surrounded the translational control of bcd. The determination of when transcription occurs for bcd could not be determined. It is known to occur early in oogenesis and localized in long before egg deposition, but they could not detect the presence of bcd in the oocyte. Furthermore, since the protein concentration was quantified using immunostain in whole mount preparations of embryos and compared against another protein called cad, which is also translated only after egg deposition is distributed evenly throughout the egg (Driever and Nüsslein-Volhard, 1988). The interaction of these proteins has biological significance but how translational control supports this has not been proven. In Figure 6, the immunostain intensity was measured against the position along the egg length. This showed that the bcdprotein concentration increases during the early hours of embryogenesis (Figure 6-E) compared to the activity of the cad protein which is distributed throughout the egg (Figure 6-D).
The protein gradients in early development are essential for patterning events that occur in the Drosophila embryo. It was discussed in previous research that bcd may have an influence over the expression of gap genes that are key dictators of the anterior/posterior axis of the embryo (Nusslein-Volhard and Wieschaus 1980). However, in addition to the control mechanisms surrounding the transcription of gap genes, bcd was also thought to interact with other maternal gene products such as cad. With the determination that bcd- embryos at early nuclear cycle 14, cad protein derived from maternal mRNA is evenly distributed as in Figure 6-D, it is suggested that the bcd protein negatively regulates cad translation or cad transcript stability directly or indirectly (Driever and Nüsslein-Volhard, 1988). In addition, another protein called hb was mentioned, that is another maternal gene that has a similar gradient in the embryo like cad, that if mutated the development of the embryo is not affected in a negative way and is large normal. This is in contrast to a mutation in bcd, which can cause significant phenotypic changes.
In textbooks, the Bcd protein serves as paradigm as the first identified morphogen, the concentration gradient of which provides the initial positional information in the anterior half of the embryo where it differentially activates segmentation genes, particularly gap genes (Spirov 2009). The activity of the morphogen was proven in this paper to be expressed in a spectacular gradient of the bcd protein, the concentration of which approximates an exponential decline with distance from the anterior pole in the nuclei of syncytial-blastoderm
embryos (Driever and Nüsslein-Volhard, 1988).
Subsequent works citing the bicoid morphogen
Evaluation of bicoid protein as a morphogen opened doorways for the developmental biologists to identify genes and mechanisms controlling the morphogenesis. Since then, a lot of research and development has taken place. Here, two significant follow researches have been picked that try to answer the queries raised on the study. After establishing the gradient of the protein, it was time for its engineering and mechanism analysis.
Bicoid is the guide to form head and thorax: Driever further extended the findings on bicoid protein confirming its essentiality for development of head and thorax in fruit flies. He with a team conducted experiments whereby purified bicoid RNA were injected in the anterior region of the early staged bicoid-deficient mutant embryos. Bcd RNA’s were substituted with 5’betaglobulin leader sequences were prepared and used for injection in mutant cells. The bcd mRNA cured the polarity of embryos by inducing gradient that led to the head formation. The location of injection became the head and rearer to that became the thorax. An interesting observation was found when bicoid RNA was injected at posterior end with the self RNA present at anterior end. It resulted in formation of two heads. It was then clearly evident that bicoid protein is translated from bicoid mRNA and can induce anterior-posterior axis without any aid from any other morphogenetic factor and is solely responsible for defining embryo sub-regions. (Driever et al. 1990).
Bicoid and Caudal role in determining anterior-posterior axis: Another aspect to study was the mechanism through which the bicoid protein determined the axis orientation in the embryo. Rivera-Pomar et al in 1996 published their findings whereby they focused on the factors along with bicoid protein role that defines the anterior pole and posterior pole. They discussed the role of bcd in establishing the concentration gradient of its posterior homeodomain counterpart, caudal (cad) protein. Bicoid protein is said to repress the posterior formation by suppressing caudal RNA translation. Caudal RNA is present all through the embryo and signifies the posterior end activating genes responsible for hindgut primordium invagination and its specification. To define anterior pole, bicoid protein binds and inhibits caudal expression in anterior region.
Current life of Christiane Nusslein-Volhard
Christiane Nusslein-Volhard continues to have major contributions in the field of developmental biology. The paper discussed, showed how the maternal gene bicoid organizes anteriorly throughout the development of the Drosophila embryo. Its mRNA is localized at the anterior tip of the oocyte and early embryo. With the discovery of bicoid as the first known morphogen it allowed for the study of embryonic pattern formation to occur and allowed other researchers in the field to have a detailed model of how a chemical morphogen could establish a gradient over a small field of cells. Pattern formation and how distinctive physiological segments are formed based on this molecule was critical to understanding. Aside from proving that bicoid was a key player in the patterning of the anterior/posterior axis. It was also demonstrated that of all the maternal effects of genes in Drosophila, only a loss of bicoid causes a complete absence of the head and thorax in mutant embryos. A researcher named Hans-Georg Frohnhofer showed that Drosophila with mutated biocid genes, organisms that wouldn’t normally develop heads or thoraxes could be completely rescued by injection of the cytoplasm containing the bicoid protein. The efficacy of these cytoplasmic rescue experiments depended on where the embryo in the cytoplasm was injected. Bicoid protein injected into the anterior pole of embryos most effectively rescued a normal phenotype, with decreasing efficacy as the injections moved towards the posterior pole. This result indicated that the anterior pole of the embryo must be the source of the bicoid protein, and as the molecule diffuses across the embryo different concentrations of bicoid control various patterns of the development of head and thoracic regions. In addition, for the first time it was proven how the bcd protein is distributed in an exponential concentration gradient with a maximum at the anterior tip, reaching background levels in the posterior third of the embryo.
Nüsslein-Volhard's later career has focused on social, ethical and philosophical issues in the sciences. She served on the German National Ethics Council from 2001 to 2006. In 2004 Nüsslein-Volhard established the Christiane Nüsslein-Volhard Foundation, which seeks to promote gender equality in science by providing support and resources to women scientists. Nüsslein-Volhard has no children, and she has said that the difficulty for women to balance research and family obligations is one of most prominent reasons women are underrepresented in leading scientific positions.
Much of the work of Nusslein-Volhard provided imperative insights into human disease. Many of the experiments that she conducted have also led to important realizations about evolution and greatly increased the scientific community's understanding of the regulation of transcription, as well as cell fate during development. She and colleague Eric Wieschaus identified the key genes responsible for embryonic development in drosophila and amassed a detailed catalog of mutations that cause physiological defects—insights that help scientists better understand human development.
Criever, W., Siegel, V., Nusslein-Volhard, C. Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen. Development, 1990. August; 109 (4): 811-20
Driever, W., Nusslein-Volhard, C. “A Gradient of bicoid Protein in Drosophila Embryos.” Cell Press. July 1st 1988. Vol. 54, 83-93.
Les Prix Nobel. The Nobel Prizes 1995, Editor Tore Frangsmyr, [Nobel Foundation], Stockholm, 1996.
Morgan, T H. Regeneration and liability to injury. Science, 1901. Aug 16;14 (346):235-48.
Nüsslein-Volhard, C. "Genetic Analysis of Pattern-Formation in the Embryo of Drosophila melanogaster." Development Genes and Evolution 183 (1977): 249–268.
Nüsslein-Volhard, C., and Wieschaus, E. "Mutations Affecting Segment Number and Polarity in Drosophila." Nature 287 (1980): 795–801.
Resnik, J., May, C., "Christiane Nusslein-Volhard (1942- )". Embryo Project Encyclopedia (2012-02-16).
Rivera-Pomar, R., Niessing, D., Schmidt-Ott, U., Gehring, W, J., Jackle, H. RNA binding and translational suppression by bicoid. Nature, 1996. Feb 22; 379 (6567): 746-9.
Spemann, H., Mangold, H. Induction of embryonic primordial by implantation of organizers from a different species. Roux’s Arch Entw Mech, 1924. 100:599–638.
Spirov, A., Khalid, F., Martina, S., Erich, F., Noll, M., Baumgartner, S. Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient”. Development 136, 605-614 (2009).
Turing, AM. The chemical basis of morphogenesis. Philos Trans R Soc Lond B Biol Sci, 1952; 237:37–72.
Wolpert, L. Positional information and the spatial pattern of cellular differentiation. J Theor Biol, 1969; 25:1–47