Talk1 Drosophila Talk 205 L

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Information about Talk1 Drosophila Talk 205 L
Health & Medicine

Published on February 6, 2009

Author: jme17

Source: slideshare.net

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Presentation of our data and results from the first antibody staining of the new allele, evePJ4 (created by Dr. Amy Bejsovec\'s lab) and the wgNE2 allele.

Characterizing eve(PJ4) Biology 205L: Experiments in Developmental Biology Fall 2006 Myung Ko, Jasmine Lau, Joshua Mendoza-Elias

Gene Regulatory Network Insert Figure 9.26 (Gilberts) runt odd TF eve wg en hh +

Consequences of wg expression Jones & Bejsovec (2003)

An anomaly: eve(PJ4)wg(NE2) Gastrulation -  wingless protein expression Cuticle patterning -  wingless expression

Gastrulation

-  wingless protein expression

Cuticle patterning

-  wingless expression

Questions: Is eve(PJ4) an amorph? Role of wg(NE2) in double mutant? Hypothesis: eve(PJ4) is an amorph wg(NE2) causes wg expression to persist

Is eve(PJ4) an amorph?

Role of wg(NE2) in double mutant?

eve(PJ4) is an amorph

wg(NE2) causes wg expression to persist

Materials and Methods Statistical Methods:  2-test, t-test Wild-type eve(3) eve(PJ4) eve(PJ4)wg(NE2) AB-staining: anti-wg antibody Stages: 9, 10, 11 Cuticle Preparations

Statistical Methods:  2-test, t-test

Part I: Is eve(PJ4) an amorph?

Are we seeing the right ratio? eve (PJ4) eve(3)  2 value = 0.333 p < 0.05  2 value= 0.022 p< 0.01 n Ratio 8 17 1 3 Mutant WT n Ratio 16 45 1 3 Mutant WT

eve (PJ4)

eve(3)

Stage 9 eve(PJ4) Mean: 5.89 (n=9) eve(3) Mean:6.21 (n=9) WT Mean: 13.2 (n= 5) P= 0.31 P= 0.00 P= 0.00

Stage 10 eve (PJ4) Mean: 5.86 (n=7) eve(3) Mean: 5.5 (n=12) WT Mean:13.5 (n= 17) P= 0.47 P= 0.00 P= 0.00

Stage 11 eve(PJ4) Mean: 5.3 (n=3) eve(3) Mean: 5 (n=3) WT Mean: 14.3 (n=3) P= 0.65 P= 0.00 P= 0.00

Mean: 5.89 Mean:6.21 Mean: 5.86 Mean: 5.5 Mean: 5.3 Mean: 5 eve(PJ4) Stage 9 Stage 10 Stage 11 eve (3) P= 0.31 P= 0.47 P= 0.65

Cuticles eve(PJ4) eve(3)

Summary eve(PJ4) behaves like an amorph As embryos mature, - decreasing # of stripes - stripes are less defined - increasing similarity b/w eve(PJ4) and eve(3)

eve(PJ4) behaves like an amorph

As embryos mature,

- decreasing # of stripes

- stripes are less defined

- increasing similarity b/w eve(PJ4) and eve(3)

Part II: Role of wg(NE2) in eve(PJ4)wg(NE2) wg(NE2) to the rescue??

Are we seeing the right ratio? eve(PJ4) eve(PJ4)wg(NE2)  2 value = 0.33 p < 0.05  2 value= 0.89 p < 0.01 n Ratio 8 17 1 3 Mutant WT 18 2 +/- n Ratio 10 8 1 1 Mutant WT

eve(PJ4)

eve(PJ4)wg(NE2)

Stage 9 eve(PJ4) Mean: 5.89 (n=9) eve(PJ4)wg(NE2) Mean:5.86 (n=7) WT Mean: 13.2 (n= 5) P= 0.95 P= 0.00 P= 0.00

Stage 10 eve(PJ4) Mean: 5.86 (n= 7) eve(PJ4)wg(NE2) Mean:5.7 (n= 10) WT Mean: 13.5 (n= 17) P= 0.76 P= 0.00 P= 0.00

Stage 11 eve(PJ4) Mean: 5.3 (n=3) eve(PJ4)wg(NE2) Mean: 6.14 (n= 7) WT Mean: 14.3 (n= 3) P= 0.12 P= 0.00 P= 0.00

Mean: 5.89 Mean:5.86 Mean: 5.86 Mean:5.7 Mean: 5.3 Mean: 6.14 P= 0.95 P= 0.76 P= 0.12 eve(PJ4) Stage 9 Stage 10 Stage 11 eve(PJ4) wg(NE2)

Cuticles eve(PJ4) eve(PJ4)wg (NE2)

Summary Effects of wg(NE2) - slower decrease in wg expression - more defined stripes - increasing deviation of eve(PJ4)wg(NE2) from eve(PJ4) - double mutant still behaves like an amorph

Effects of wg(NE2)

- slower decrease in wg expression

- more defined stripes

- increasing deviation of eve(PJ4)wg(NE2) from eve(PJ4)

- double mutant still behaves like an amorph

Discussion EVX1, a human homeobox gene homologous to eve - Transcriptional repressor: specifies A-P axis in embryos - EVX1 -/- : increased oncogenic potential

EVX1, a human homeobox gene homologous to eve

- Transcriptional repressor: specifies A-P axis in embryos

- EVX1 -/- : increased oncogenic potential

Future Directions 0-4 hours Ab staining 0-4 hours and 4-8 hours In Situ hybridization eve(1) mutants Stages 12 to 15 (cuticle secretion) Sequence eve(PJ4)

0-4 hours Ab staining

0-4 hours and 4-8 hours In Situ hybridization

eve(1) mutants

Stages 12 to 15 (cuticle secretion)

Sequence eve(PJ4)

Acknowledgements Dr. Bejsovec Dr. Perz-Edwards Wendy Beane Monica Zhang Our wonderful classmates!

Dr. Bejsovec

Dr. Perz-Edwards

Wendy Beane

Monica Zhang

Our wonderful classmates!

Thank you!

References Paul M. Mcdonald, Phil Ingham and Gary Struhi. Isolation, Structure, and Expression of even-skipped: A Second Pair-Rule Gene of Drosophila Containing a Homeo Box . Cell: Vol. 47, 721-734 (1986) Marcel van den Heuvel, Roel Nusse, Paul Johnston, and Peter A. Lawrence. Distribution of the wingless Gene Product in Drosophila Embryos: A Protein Involved in Cell-Cell Communication . Cell: Vol. 59, 739-749 (1989) Herman A. Dierick and Amy Bejsovec. Functional analysis of Wingless reveals a link between intercellular ligand transport and dorsal-cell-specific signaling. Development: 125, 4729-4738 (1998) 4. Whitney M Jones and Amy Bejsovec. Wingless Signaling: An Axin to Grind. Current Biology: Vol.13, R479-R481 (2003)

Paul M. Mcdonald, Phil Ingham and Gary Struhi. Isolation, Structure, and Expression of even-skipped: A Second Pair-Rule Gene of Drosophila Containing a Homeo Box . Cell: Vol. 47, 721-734 (1986)

Marcel van den Heuvel, Roel Nusse, Paul Johnston, and Peter A. Lawrence. Distribution of the wingless Gene Product in Drosophila Embryos: A Protein Involved in Cell-Cell Communication . Cell: Vol. 59, 739-749 (1989)

Herman A. Dierick and Amy Bejsovec. Functional analysis of Wingless reveals a link between intercellular ligand transport and dorsal-cell-specific signaling. Development: 125, 4729-4738 (1998)

4. Whitney M Jones and Amy Bejsovec. Wingless Signaling: An Axin to Grind. Current Biology: Vol.13, R479-R481 (2003)

5. Antonio Faiella, Maurizio D'Esposito, Marco Rambaldi, Dario Acampora, Silvia Balsofiore, Anna Stornaiuolo, Antonio Mallamaci, Enrica Migliaccio, Massimo Gulisano, Antonio Simeone and Edoardo Boncinelli. Isolation and mapping of EVX1, a human homeobox gene homologous to even-skipped, localized at the 5' end ofHOXi locus on chromosome 7. Nucleic Acids Research: Vol. 19, No. 23 6541-6545 (1991). 6. Chi Li and James Manley. Even-skipped Represses Transcription by Binding TATA Binding Protein and Blocking the TFIID-TATA Box Interaction . Molecular and Cellular Biology: Vol. 18, No.7, p. 3771–3781 (1998). 7. Paola Briata, Rinke Van De Werken, Irma Airoldi, Cristina Ilengo, Erica Di Blas, Edoardo Boncinelli, and Giorgio Corte. Transcriptional Repression by the Human Homeobox Protein EVX1 in Transfected Mammalian Cells. The Journal of Biological Chemistry: Vol . 270, No. 46, pp. 27695–27701, (1995).

5. Antonio Faiella, Maurizio D'Esposito, Marco Rambaldi, Dario Acampora, Silvia Balsofiore, Anna Stornaiuolo, Antonio Mallamaci, Enrica Migliaccio, Massimo Gulisano, Antonio Simeone and Edoardo Boncinelli. Isolation and mapping of EVX1, a human homeobox gene homologous to even-skipped, localized at the 5' end ofHOXi locus on chromosome 7. Nucleic Acids Research: Vol. 19, No. 23 6541-6545 (1991).

6. Chi Li and James Manley. Even-skipped Represses Transcription by Binding TATA Binding Protein and Blocking the TFIID-TATA Box Interaction . Molecular and Cellular Biology: Vol. 18, No.7, p. 3771–3781 (1998).

7. Paola Briata, Rinke Van De Werken, Irma Airoldi, Cristina Ilengo, Erica Di Blas, Edoardo Boncinelli, and Giorgio Corte. Transcriptional Repression by the Human Homeobox Protein EVX1 in Transfected Mammalian Cells. The Journal of Biological Chemistry: Vol . 270, No. 46, pp. 27695–27701, (1995).

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