INFORMATION FOR THE DORSAL-VENTRAL PATTERN OF DROSOPHILA EMBRYO IS STORED AS MATERNAL �m-RNA

SUBMITTED TO ,

DR. P. VARALAKSHMI

DR. B. ASHOKKUMAR

SUBMITTED BY ,

TANDRA SANPUI

RITHIKA NADAR

RIDDHIMAN CHATTERJEE

CONTENT

• History
• Overview of the paper
• Proteins in signaling cascade.
• Methods
• Dosage response
• Conclusion
• Reference

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Kathryn Virginia Anderson�(1952-2020)

• Education:

schooling from Point Lama High School.

graduated from the University of California, Berkeley in Biochemistry.

In 1973, she began her post-graduate studies in neurodevelopment at Stanford university.

In 1977, she opted for doctoral studies in the field of genetics with Judith Lengyel at the University of California.

Received her Ph.D. degree in 1980.

• Award:

She was elected to the National Academy of Science in 2002.

 Thomas Hunt Morgan Medal in 2012 by Genetics Society of America.

The Edwin Grant Conklin Medal by the Society for Developmental Biology in 2016.

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Anderson said,’Beautiful embryos� Full of life and mysteries� You and I can solve’

Judith Lengyel

(1945-2004)

Research Work

• In 1981, Anderson moved to the Friedrich Miescher Laboratory in Tübingen, Germany, to work with Christiane Nüsslein-Volhard.
• Between 1984 and 1986, Anderson and Nüsslein-Volhard coauthored five publications in which they identified genes involved in components of the dorsal-ventral signaling, such as Toll, easter, tube, Pelle, spätzle, pipe, nudel, and snake.
• From 1993-1994, she carried out research on mouse embryonic at the National Institute for Medical Research, the united kingdom under the guidance of Rosa Beddington.
• In 1996, she joined the Molecular Biology Program at the Memorial Sloan Kettering Cancer Centre, New York.
• In 2003, using forward genetics, they discovered a previously unknown role for cilia, a microscopic, hair-like structure found on the surface of certain cells, in receiving signals from a protein called Hedgehog. 

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Rosa Beddington

(1956-2001)

Christiane Nüsslein-Volhard

• Born: 20 October 1942, Magdeburg, Germany.
• She is the only woman from Germany to have received a Nobel Prize in the sciences.
• Awards: The Nobel Prize in Physiology or Medicine 1995 with Eric Wieschaus and  Edward B Lewis.
• Albert Lasker Medical Research Award, New York(USA).
• Affiliation at the time of the award: Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany
• Prize motivation: “for their discoveries concerning the genetic control of early embryonic development”
• Education:
• Diploma degree in Biochemistry in 1969.
• Doctorate in Tubingen University in 1973 and studied about DNA-Protein interaction, the binding of RNA Polymerase in E. coli.
• Post-doc. in Basel and Freiburg in the year 1975 and 1977.
• From 1978-1980, Head of Group, European Molecular Biology Laboratory, Heidelberg.
• Head of a junior research group at the Friedrich Miescher Laboratory of the Max Planck Society in Tubingen(1981-1984).
• In 1985, director of Max Planck Institute.

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Photo from the Nobel Foundation archive.

Research Work

• When a fertilized egg divides and forms new cells which, in turn, also divide. Initially, these cells appear identical, but in time, they begin to change. Some cells go to make up the heart, others nerve paths, and still others strands of hair, for example, Genes regulate this process. Christiane Nusslein-Volhard and Eric Wieschaus studied the development of fruit flies and, around 1980, succeeded in identifying and classifying the 15 genes that direct the cells to form a new fly.

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• In 1975, she started to research flies in Walter Gehring’s lab on bicaudal.

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• In 1977, she worked in Klaus Sander’s law with Margit Schardin in Freiburg.

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• In 1978, she worked with Pedro Santamaria to rescue the dorsal phenotype by transplantation of wild-type cytoplasm.

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• In 1978-1980, she worked with Eric on Kruppel.

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Eric Wieschaus

Edward B Lewis

Cont…..

• In 1981, she joined in Friedrich-Miescher-Laboratorium and Kathryn joined as a postdoc.

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• Anderson studied dorsal genes and pursue rescue experiments.

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• Gerd Jurgens and Volhard almost finished the research on segmentation mutants in 1984.

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• Trudi Schupbach and Eric Wieschaus had isolated maternal mutants.

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• An important method to analyze the function of the genes, they used in my laboratory was cytoplasmic transplantation.

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Trudi Schupbach

Friedrich-Miescher-Laboratorium

• The Friedrich Miescher Laboratory (FML) of the Max Planck Society is a biological research institute located on the Society's campus in Tübingen, Germany, named after Friedrich Miescher, founded in 1969.
• Offer highly qualified junior scientists in biology an opportunity to establish independent research groups and pursue their own line of research within a five-year period.

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Max Planck Society

• The Max Planck Society for the Advancement of Science is a formally independent non-governmental and non-profit association of German research institutes.
• Founded in 1911 as the Kaiser Wilhelm Society.
• it was renamed the Max Planck Society in 1948 in honor of its former president, theoretical physicist Max Planck.
• The society is funded by the federal and state governments of Germany.
• According to its primary goal, the Max Planck Society supports fundamental research in the natural, life, and social sciences, the arts, and humanities in its 86 (as of December 2018) Max Planck Institutes.

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Max Karl Ernst Ludwig Planck(1858-1947)

Let’s go into the past discovery….

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Partial rescue of dorsal, a maternal effect mutation affecting the dorsoventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm.

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Pedro Santamaria and Christiane Nusslein- Volhard

12 July ,1983

DOI: 10.1038/311223a0�Publication date: 1984 Sep 20(Nature publication)�

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OVERVIEW

• This paper demonstrated that early dorsal-ventral pattern information is stored as maternal mRNA in flies and devised the method of identifying genes encoding such genes.
• They identified the individual genes which are responsible for dorsal-ventral pattern formation in Drosophila, but they wanted to know individual gene functions. Dorsal-most cells are produced in the absence of any one of these gene functions indicating that each one has an essential component of one pattern-forming process.
• They used a rescue assay for demonstrating the information from several dorsalizing maternal-effect genes is found in the wild-type egg in the form of poly(A) RNA.

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Proteins involved in the signaling cascade

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Signalling Cascade

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Methods

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Interpretation

• Injection of wild type cytoplasm into easter, tube & snake Drosophila mutants.
• Cuticle preparation of uninjected easter, tube & snake embryos all show the same tube like form of dorsal cuticle. Fig (a) uninjected embryo.
• In Fig (b) easter responds most weakly to injection developing only filzkörper & narrowed rows of ventral denticles.
• In Fig (c) tube responds more strongly develops ventral denticles in addition to filzkörper.
• In Fig (d) snake embryos show a total restoration of dorsal ventral pattern element after injection.

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Whether snake rescuing is rna or not ?

• Cytoplasmic RNA from wild type embryos was injected into snake embryos.
• The injection of RNA dramatically rescued the snake phenotype.
• The injected embryos developed filzkörper & ventral denticle bands.
• It was observed that it is not due to some non RNA contaminant.
• The rescuing activity is protease insensitive, RNase sensitive & alkali sensitive.

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Whether the rescuing rna is mRNA?

• The cytoplasmic RNA was separated on oligo (dT)- cellulose into poly(A)+ and poly(A)- fractions, it was tested for rescuing activity.
• The rescuing activity was recovered in the poly(A)+ fraction, the snake- rescuing RNA is therefore polyadenylated and is mRNA.
• Cuticle of snake embryo was injected with Poly (A)+ RNA isolated from a wild type embryo showing full dorsal ventral pattern.

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CHARACTERISTICS OF RESCUE BY CYTOPLASM AND RNA FOR SNAKE, EASTER AND TUBE

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CHARACTERISTICS OF RESCUE BY CYTOPLASM AND RNA FOR SNAKE, EASTER AND TUBE

• Of the three loci studied, snake responds most strongly to injection of both cytoplasm and RNA.
• The easter rescuing RNA apparently differs from both tube and snake in its degree of polyadenylation. The poly (A)­­- RNA fraction rescues easter only slightly more weakly than total RNA (data not shown).
• The concentrations of the poly (A)+ RNAs that rescue snake, tube and easter, all decrease rapidly during early development. The tube and easter rescuing activities are barely detectable in poly(A)+ RNA isolated from 3-7h embryos, and the activity of snake is also reduced.
• For tube, exceptionally, cytoplasm from extended germ band stage donors rescue as effectively as cleavage cytoplasm, although the RNA isolated from that stage doesn’t rescue significantly.

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DOSE RESPONSE RELATIONSHIP FOR RESCUE OF SNAKE BY POLY (A)+ RNA

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CONT….

• The phenotypic response seen depends on the concentration of the injected RNA. Injection of high concentrations of poly (A)+ RNA restores the complete range of dorsal-ventral pattern elements in snake embryos.

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• As the concentration is decreased, most of the rescued embryos have either clearly narrowed ventral denticle belts or develop only filzkörper.

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• Another drop in concentration produces embryos which do not form ventral denticles, but still developed filzkörper.

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CONT…

• Thus the highest degree of ventralness achieved is proportional to the concentration of rescuing RNA , with a continuous increase in the quantitative nature of the response over a 50- fold range of RNA concentration.

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CONCLUSION

• The maternal effect mutations , described here, each remove a single, genetically defined component required in embryonic pattern formation. The phenotypic rescue of these mutants by injection provides a specific assay to probe the molecular basis of embryonic dorsal-ventral pattern formation. As poly (A)+ RNA isolated from cleavage wild type embryos, rescue snake, tube and easter, thus these RNAs are a normal storage form of morphogenetic information from these genes.
• The dose response relationship between the amount of RNA injected and the phenotypic response of snake embryos shows that the level of ventralness achieved in the embryo is a direct, continuous function of the amount of snake rescuing RNA present in the embryo.
• The rescue response of tube and easter show analogous dose-response relationship, although more amount of RNA is required for the same level of response.
• Thus, snake, easter and tube are all essential components of a system of graded, positional information.
• The cytoplasmic rescuing activity of each gene has unique pattern , which reflects their unique roles in the establishment of dorso-ventral pattern.
• The demonstration that a group of morphogenetic mRNAs is present in early wild type embryos suggests that a control of translation of these mRNAs may be a significant aspect of the mechanism of dorso ventral pattern formation.

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After this discovery….

F Müller Holtkamp

&

Herbert Jäckle

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Spätzle embryos also respond weakly to cytoplasmic injection but are nevertheless rescuable by RNA.

Reference

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• Information for the dorsal--ventral pattern of the Drosophila embryo is stored as maternal mRNA Anderson KV and Nüsslein-Volhard C; Nature. 1984 Sep 20-26;311(5983):223-7
• Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster.Gans M, Audit C, Masson Mgenetics, (4):683-704 1975MED: 814037
• Developmental genetics of the Drosophila egg. I. Identification of 59 sex-linked cistrons with maternal effects on embryonic development.Mohler JDGenetics, (2):259-272 1977MED: 405273
• Nüsslein-Volhard, C. in Determinants of Spatial Organization (eds Subtelney, S. & Konigsberg, I. R.) 105–211 (Academic, New York, 1979).
• Partial rescue of dorsal, a maternal effect mutation affecting the dorso-ventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm.Santamaria P, Nusslein-Volhard CEMBO J, (10):1695-1699 1983MED: 16453473
• A dorso-ventral shift of embryonic primordia in a new maternal-effect mutant of Drosophila.Nusslein-Volhard C, Lohs-Schardin M, Sander K, Cremer CNature, (5746):474-476 1980MED: 6766208
• Rates of synthesis of major classes of RNA in Drosophila embryos.Anderson KV, Lengyel JADev Biol, (1):217-231 1979MED: 110635

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Thank you