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Introduction

Eric Wieschaus's and Christian Nusslein-Volhard's experiments on the developmental patterning of Drosophila melanogaster are some of the most important experiments in the history of developmental biology. By performing genetic screens on fruit flies, they successfully identified several mutations that caused malformations in embryonic development and are therefore associated with genes that are essential for this development. This page is intended to summarize their experiments and discuss the way in which their findings have impacted the contemporary scientific community.

Background

During Drosophila development cellular membranes do not form until after the thirteenth nuclear division, therefore all the dividing nuclei share a common cytoplasm where materials can diffuse throughout the whole embryo (Akam, M., 1987). In Drosophila embryos the specification of cell types along the anterior-posterior and dorsal-ventral axes is accomplished by the interactions of cytoplamic materials within the single multinucleated cell. The maternally acting genes fall into three classes having effects on three distinct regions of the embryo. The torso group causes defects in the terminal regions of the embryo (Akam, M., 1987). The bicoid genes affect the anterior of the Drosophila embryos while the oskar genes, affect the posterior parts(Akam, M., 1987). Transplant experiments were used to show that these genes are localized at the anterior and posterior poles of the embryo and that their influence spreads into the more central regions (Frohnhofer & Nusslein-Volhard, 1986). An example of how these genes affect the zygotic genes can be seen with the Bicoid genes. Bicoid contains a homeobox sequence so the protein may act as a transcription factor. The mRNA is localized at the anterior pole of the egg and the message is translated early in the development of the blastoderm and the protein diffuses forming a gradient from anterior to posterior (Frigerio et al, 1986). Zygotic genes interact with maternal proteins by responding to and elaborating the information set up by the maternal genes (Nusslein-Volhard & Driever, 1988). Three phenotypic classes of zygotic segmentation genes that interact with maternal proteins have been defined by Nusslein-Volhard & Wieshaus (1980), segment-polarity, pair-rule, and gap. The 1995 Nobel Prize in Physiology or Medicine was awarded jointly to Edward B. Lewis, Christiane Nüsslein-Volhard and Eric F. Wieschaus "for their discoveries concerning the genetic control of early embryonic development" (Nobel, 2013).

Maternal effect

Maternal effect genes produce messenger RNAs that are placed in different region of the egg. These messages encode transcriptional and translational regulatory proteins that diffuse through the blastoderm and activate or represses the expression of certain zygotic genes. The first zygotic transcription factors that are activated are the gap genes. These genes are expressed in particular wide and overlapping domains and define broad territories of the embryo. These gap genes encode transcriptional factors that regulate the transcription of pair-rule genes which divide the embryo into metameric units. The transcription of the different pair-rule genes result in a striped pattern of seven transverse bands that run perpendicular to the anterior-posterior axes. The proteins encoded by the pair-rule genes are transcription factors that activate the segment polarity genes, whose mRNA and protein products divide the embryo into 14 segment-wide units, establishing the metameric nature of the embryo (Wieschaus, E, 1984). At the same time the protein products of the gap, pair-rule, and segment polarity genes interact to define the spatial domains of the homeotic genes that define the identities of each of the segments, the homeotic selector genes transcription determines the developmental fate of each segment. The segments that are produced by the segment polarity genes are all different but have certain morphological features in common. The larval body is composed of three thoracic and eight abdominal segments. The anterior of each segment is marked with a band of denticles, most of which point posteriorly. The posterior part of each segment is naked. The segment borders run along the anterior margins of the denticle bands. the denticles themselves differ in width, in the thoracic segments the bands are narrow with fine denticles whereas those in the abdominal segments are broader and comprised of thick pigmented denticles. The work of Nusslein-Volhard and Wieschaus looks for mutations that affect the segmental pattern within the zygotic genome. They found 15 loci which show one of the three novel types of pattern alterations: pattern duplication in each segment (segment polarity mutants), pattern deletion in alternating segments (pair-rule mutants), and deletion of a group of adjacent segments (gap mutants). The work by Nusslein-Volhard and Wieschaus identified a hierarchy of genes that establish anterior-posterior polarity, and divide the embryo into a specific number of segments with different identities.

Segment Polarity mutations

Homozygous mutations in the segment polarity genes result in zygotic lethals in which the phenotypes have the normal number of segments yet within each segment a defined fraction of the normal pattern is deleted. The remaining portion is present as a mirror-image duplication. Six loci were identified in the original Nusslein Volhard and Weischaus paper, namley fused, wingless, cubitus interruptis, gooseberry, hedgehog, and patch. In all mutants, except patch, the region deleted includes the naked posterior part and the duplication involves a fraction on the anterior denticle band. In mutant larvae, the ventral side of each segment is almost entirely covered with denticles. The denticles of the abdominal segments are larger and pigmented and the denticles of the thorax are short and pale. In fused and gooseberry mutants the anterior margin of the region is duplicated and coincides with the segment boundary in both cases. In wingless and hedgehog it lies posterior to the boundary. The duplicated region for patch mutants have a duplicated region that includes some naked cuticle anterior to each denticle band. It is suggested, from these mutants, that these loci are involved in the specification of the basic pattern of the segmental units.

Pair-rule mutants

In the work by Nusslein-Volhard and Wieschaus, six loci were identified that when mutated resulted in the deletion of a part of the cuticle pattern in every other segment. Each of the six loci is characterized by its own specific pattern of deletions. In even skipped larvae, for example, the denticle bands and adjacent naked cuticle of the pro- and metathoracic, and the 2nd, 4th, 6th, and 8th abdominal segments are lacking. This results in larvae with half the normal number of denticle bands separated by enlarged regions of naked cuticle. In paired mutant larvae the reduction in segments results in the deletion of the naked posterior part of the odd-numbered segments and the anterior denticle bands of the even-numbered segments.

Gap mutations

A mutation of the gap genes results in whole segments of the embryo being alerted. The first two classes of gap genes causes an alteration where the pattern is repeated at specific intervals along the antero-posterior axis of the embryo. In the third class a single group of up to eight adjacent segments are deleted from the final pattern. In the original paper three loci were found that cause such gaps, they were Kruppel, knirps, and hunchback. The effect of mutations in these genes causes gaps in specific regions of segmentation, for example, when an embryo is homozygous for the Kruppel mutation, it lacks thorax and anterior abdomen.

Methods/Techniques/Data

Mutagenizing flies- Flies were treated with a mutagenic substance that would cause mutations in approximately half of the genes. This is a random process, it doesn’t target specific genes. Thus, they randomly mutated the genes and then screened for a particular phenotype. In this case they looked for mutations that disrupted the pattern of development of the embryo. They used the cuticle of the larvae that is produced at the end of embryogenesis to assess the patterning of structures along the anterior posterior axis. The identified mutations were then put into three phenotypic classes based on similarity.

Analysis of results- in analyzing the results of the mutations, they were able to find 15 genes that were associated with mutations in segmentation. The 15 genes that they found using saturation mutagenesis were cubitus interruptus, wingless, gooseberry, hedgehog, fused, patch, paired, even-skipped, odd-skipped, barrel, runt, engrailed, Kruppel, knirps and hunchback (Moran, 2008).

Establishing fly lines- Genetic screens were used to find mutations within the gene that affected biological processes. The genes they were looking for were recessive, so in order to see the recessive trait emerge in the population they had to do multiple genetic crosses to establish fly lines that were homozygous recessive for the mutation. To do this, they first made mutations within the population. Secondly, they started producing inbred flies to get a specific trait by crossing flies within the same family. Since these flies were now specific for a trait, they then picked out the females and mated them to males that had been fed mutagenic agents, and the progeny (F1) would be heterozygous for the traits. They then mated the heterozygous males with heterozygous females from the same family again to get homozygotes.

Screening of results- Using these heterozygous stock lines to generate homozygous mutant embryos, they were able to determine whether a mutation was gap, segment polarity or pair-rule (using, 2014). They determined this by examining the cuticle at the end of embryogenesis. Particularly, they looked at the denticles on the cuticle. Flies that had mutations in every segment were placed in one category, mutations that were seen in every other segment were placed in another, and mutations observed in just one particular group of segments were placed in another. By this method they could analyze the genes in each category of mutations by crossing the mutant flies with flies that had other known mutations in order to map the genes. They could also screen the mutations against wildtype flies to assess which genes were effected by the mutations.


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Relevance

Through these experiments, Weischaus and Nusslein-Volhard were able to categorize developmental mutations in Drosophila into three groups: segment polarity mutations, pair-rule mutations, and gap mutations. The defining characteristics of these groups lead to certain conclusions about the entire development of the organism.

Segment polarity mutations cause deletions of the posterior halves of each segment pattern and a subsequent mirroring of the remaining anterior half patterning into the posterior region of each segment. The experimenters theorized that this is because the loci associated with segment polarity mutants are important in developing the basic patterning of the segments in the embryo. Essentially, these loci ensure that each segment will have an anterior and posterior pattern that is repeated through the embryo.

Pair-rule mutations cause deletions of patterning in every other segments, therefore seven of the fourteen segments were effected. This repetition of missing patterns implies that the embryo is aligned during development in a series of repeating patterns that are grouped in pairs.

Gap mutations cause deletions of entire groups of segments, creating a ‘gap’ in the patterning of the embryo. The resulting fly would have a shorter body, as if a portion of it had been cut and removed and the remaining portions stitched back together. Unlike segment polarity mutations and pair-rule mutations, this mutation does not affect the embryo in a repeated pattern, suggesting that the loci that cause these types of mutations are involved in processes that do not occur in any spatial pattern or repetition.

Application to Medicine
By elucidating the different levels of developmental organization and patterning in fruit flies, this work was a landmark study in the field of developmental biology. These insights about patterning and development could be projected from fruit flies to many organisms, including humans. The three groups of loci have homologues in humans, which allows the projection of these data to provide explanations for human development. Birth defects in humans are thought to be greatly affected by these Drosophila patterning homologues (Using lethal 2014), so a deeper understanding of the processes controlled by these homologues is essential for preventing and treating birth defects. By understanding what genes control development, researchers can look for causes of their mutations and eliminate the chance of certain severe birth defects. In particular, segment polarity genes encode paracrine factors, or ligands, such as hedgehog (hh) and wingless (wg) that cannot be produced when the gene is mutated, and therefore the receptors that these ligands bind to are never activated. In the case of segment polarity genes, this causes developmental abnormalities due to the targets of the mutated gene never being activated. Examples of the birth defects that can occur from the inactivation of the hedgehog pathway are Pallister Hall syndrome, polydactyly, and poor development of the hypothalamus and pituitary gland. The hedgehog paracrine factor is the ligand involved in the Hedgehog signal transduction pathway. In the absence of hh, the patched protein receptor tethers the Ci protein to the microtubules and the binding of other proteins to Ci allows Ci to be cleaved into a transcriptional repressor that represses transcription of a gene (Roessler et al., 1994). Yet when hh binds to the patched receptor there is a conformational change that allows the release of the tethered Ci protein allowing the whole Ci protein to enter the nucleus and act as a transcriptional activator of essential cell growth genes (Roessler et al., 1994). Over activation of the hedgehog pathway, however, results in constant stimulation of these genes and results in unrestricted cell growth, causing basal cell carcinomas and tumors of the basal cell layer of the epidermis. This shows that proper regulation of these developmental genes are not essential just for initial embryonic development, but they are also essential for the survival of the organism further into its life cycle. Armed with this knowledge, drug companies now have a clearer picture of what is causing certain cancers and can theoretically tailor treatments to compensate for an overactive hedgehog pathway.

Application to Other Research
Understanding what causes malformed embryos induces, to some degree, an inherent understanding of what it takes to produce a successful embryo and eventual healthy organism. If researchers are able to control the development of organisms at a fine level, the chances of the survival of the organism they are studying can be increased dramatically, leading to more successful and efficient research in general. Furthermore, revealing some of the puzzle pieces that are needed to form an embryo from scratch brings the scientific community one step closer to efficiently generating clones of individual organisms. Having these puzzle pieces in mind during cloning experiments would be a crucial factor to the success of the clones, thereby raising the possibility of generating clones in a consistent fashion.

Reference

Akam, M. (1987). The molecular basis for metameric pattern in the Drosophila embryo. Development 101, 1-22

Driever, W. & Nusslein-Volhard, C (1988). The bicoid protein determines position in the Drosophila embryo in a concentration dependent manner. Cell 54, 95-104.

Frigerio, G., Burri, M., Bopp, D., Baumgarther, S. & Noll, M. (1986). Structure of the segmentation gene paired and the Drosophila prd gene ser as part of a gene network. Cell 47, 735-756.

Frohnkofer, H.G. & Nusslein-Volhard, C. (1987). Maternal genes required for the anterior localization of bicoid activity in the embryo of Drosophila. Genes Dev. 1, 880-890.

Genetic Cross. Green Facts Facts on Health and the Environment. GreenFacts 2001–2013 GreenFacts®, 2013. http://www.greenfacts.org/glossary/ghi/genetic-cross.htm. 6 February 2014.

Genetic Screening. TheFreeDictionary. Random House Kernerman Webster's College Dictionary, © 2010 K Dictionaries Ltd. Copyright 2005, 1997, 1991 by Random House, Inc. All rights reserved, 2014. http://www.thefreedictionary.com/genetic+screening. 6 February 2014.

Moran, Laurence A. Nobel Laureates: Christiane Nüsslein-Volhard and Eric Wieschaus. Sandwalk Strolling with a skeptical biochemist, 2008. http://sandwalk.blogspot.com/2008/10/nobel-laureates-christiane-nsslein.html. 6 February 2014.

Nusslein-Volhard, C., Frohnhofer, H. G. & Lehmann, R. (1987). Determination of anterior posterior polarity in Drosophila. Science 238, 1675-1681.

Nusslein-Volhard, C. & Wieschaus, E. (1980). Mutations affecting segment number and polarity in Drosophila. Nature, Lond. 287, 795-801.

Nobel Media AB (2013). The Nobel Prize in Physiology or Medicine 1995. Nobelprize.org.

Rousseau, F., Bonaventure J., Legeai-Mallet L.,Pelet A., Rozet, Maroteaux P., Merrer, and Munnich A. 1994. Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 371:252-254.

Using Lethal Mutation to Study Development. Classica Experiment 9.5. http://basic.shsmu.edu.cn/jpkc/cellbiota/resource/exper/10.pdf. 6 February 2014.

Wieschaus, E., Nusslein-Volhard, C. & Klunding, H. (1984). Kruppel, a gene whose activity is required early in the zygotic genome for normal embryonic segmentation. Devl Biol. 104,172-186


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