Tribolium embryology

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Tribolium embryology
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Why Tribolium castaneum ???

• provides context for understanding the
  relationship between genetic and morphological
  change
• offers easy molecular analysis and molecular
  embryology
• has a short developmental period

Tribolium castaneum
Red Flour Beetle
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Goals

• To establish methods for studying embryogenesis
  in Tribolium castaneum and develop a working
  understanding of the Tribolium embryo
• Study the expression and regulation of genes
  important in the development of the embryo

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Preparation

• In order to analyze the Tribolium embryo egg
  collections were made every 12 hours for about a
  week
• allowed for a wide range of embryo stages to be
  studied
• embryos stored until needed

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Morphology

• Embryos were fixed to preserve morphological
  integrity
• Embryos were embedded in paraffin wax, sectioned
  and mounted on microscope slides
• Slides were stained using Harris Haematoxylin,
  Bierbrich Scarlet/Orange II, and Fast Green

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Morphology Microscopy

• Sections were analyzed using various microscopic
  techniques including DIC, brightfield
  illumination, and darkfield illumination
• Photographs of embryo morphology were obtained
  using Snappy software and edited using Adobe
  Photoshop

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Early Embryo Development
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Middle Embryo Development
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Late Embryo Development
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Expression and Regulation of Developmental Genes

• PTL and UTX genes were analyzed
• PTL expressed in the thorax and UTX expressed in
  the abdomen of the developing embryo
• SP6 polymerase and template DNA used to generate
  anti-sense RNA
• In Situ Hybridization- antisense RNA anneals to
  mRNA target
• Antibody binds to anti-sense RNA allowing for
  detection

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Analysis
PTL
UTX
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FUTURE DIRECTIONS

• Continue looking at gene expression and
  regulation of PLT and UTX via in situ
  hybridization
• Explore new fixation methods
• different methods for different developmental
  stages
• Construct new cDNA libraries
• Analyze other developmental genes