An Analysis of Zebrafish Chromosomes and Development

1
An Analysis of Zebrafish Chromosomes and
Development

• S. Torrealday, D. Greene, G. Packert, T. Petrino
  and Y-W.P. Lin
• Barry University, Miami Shores, FL

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General Goals

• To identify and characterize the Polycomb gene in
  zebrafish.
• To understand the significance of the Polycomb
  gene product in zebrafish development

3
Zebrafish Life Cycle
4
Similarities in Vertebrate Embryos
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Polycomb in Development

• Gene is highly conserved from insect to man
• One of about 40 genes within the Pc group
• Pc proteins associate with DNA and silence
  transcription
• Regulate Homeotic gene expression
• Essential genes
• Loss causes lethality in embryos
• Loss in adults associated with malignancies

6
Homeotic Gene Mutations
7
Model for Polycomb repression of Homeotic genes
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Specific Goals

• To prepare chromosomes for karyotyping
• To determine the presence of Polycomb gene
  product during oogenesis and early embryogenesis
  in zebrafish using antibody staining

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Materials and Methods

• Isolation of zebrafish chromosomes
• Antibody staining of zebrafish ovaries
• Antibody staining of unfertilized zebrafish eggs
  and early embryos

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Marble Breeding
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Chromosomal Isolation
o
o
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Antibody Staining
o
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Results

• Karyotyping of zebrafish chromosomes

x400
14

• Karyotype alignment

X 400
15

• Antibody staining of oocytes

x40
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Zebrafish Embryo
x400
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Genetic Hierarchy in Development

• Maternal Effect genes
• Segmentation genes
• Gap genes
• Pair rule genes
• Segment polarity genes
• Homeotic genes

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Pc Stained Embryos(128 cell stage)
x200
x100
19
Pc Stained Embryos(18 Hr)
x200
x100
20
Pc Stained Embryos(24 Hr)
x100
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Future Goals

• Further evaluate Polycomb distribution during
  embryogenesis
• Identify Polycomb protein binding sites on the
  Zebrafish chromosomes
• Study the interactions between the Polycomb gene
  and Homeotic genes during development

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Summary

• Polycomb product
• Expressed in oogenesis
• Not Expressed in embryogenesis (prior to 128 cell
  stage)
• Expressed in embryogenesis (after 128 cell stage)
• Follows the patterns in Drosophila and mice
• Zebrafish is a good model for the study of
  Polycomb in development

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Acknowledgements

• Dr. Renato Paro, University of Cologne, Germany
• Celeste Nadal
• Sr. John Karen Frei
• NIH MBRS SCORE /Barry University Research
  Scholarship Grants

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Questions?