Transcript Time Lapse Imaging of Zebra fish Embryonic Development and

Cell Division Dynamics
A Closer Look at Gastrulation in Zebrafish
Salome Chuang, Daniel Holland-Moritz
Mentor: Luca Caneparo
Summer Research Connection
Oak Crest Institute of Science
California Institute of Technology
July 30, 2010
Cell Division: A Crucial Biological
Process
• Normal:
• Growth
• Development
• Tissue repair
• Abnormal:
• Cancer
http://tainano.com/chin/Molecular%20Biology%20Glossary.htm
An Introduction to Cell Division
Our Project: Dynamics of Cell
Division in Embryos
End of Cell Division
Beginning of Cell Division
Spindle Poles
Difficulties of Studying Cell Division
in Live Embryos
• Ethical concerns (humans)
• Opaque embryos
• Development Rate
• Production Rate
• Manipulation
• Storage/Care
• External vs. Internal
Development
Images from Kidscientist.com and odec.ca
Zebrafish: An Ideal Model for
Studying Embryonic Cell Division

Very common for use in research because of:

Rapid reproductive rate

Transparent embryos

Rapid development

Easily cared for and contained

Easy to induce and screen for mutations

Genome almost fully sequenced
Bar: 250 µm
Images from focusonnature.be
A Brief History on Zebrafish


George Streisinger
Established zebrafish as an ideal subject for imaging and
manipulation.

Very small species of minnow

Native to the Ganges region of India

Named for distinctive stripes

Since then zebrafish have been popularized as a model for
studying vertebrate development:

Cell biology and development

Organ system development

Genetic studies
Image from www.zfin.org
Stages of Zebrafish Development
Key:
=Injection stage
=Imaging stage
Shield Stage to Gastrulation
Cell Division in Dorsal Region of
Embryos
Dorsal
Posterior
Significance of the Dorsal Region

Dorsal region becomes...


Nervous system
Application:


Cell orientation could have an impact on brain and
nervous system development at an early age
Cell imaging provides better insight into....


Embryonic development
Cell division mechanisms and signaling in vertebrates
Shield Stage to Gastrulation: Trends

Tissue differentiation


Dorsal region: Definite pattern
Cells divide along the anterior-posterior plane
Concha, Adams, 1998
Visualizing Cell Division Dynamics
End of Cell Division
Beginning of Cell Division
Spindle Poles
Labeling of Cell Structures to Study
Cell Division Dynamic
Red: Nucleus
Green: Cytoskeleton
Methods
Genetic Engineering of Fluorescent Embryos

mRNA codes for fluorescent
protein in cytoskeleton and
nucleus
DNA  RNA  Protein


Synthetic mRNA produced
for injection
mRNA injected into embryos
at one cell stage
Image from protein-dynamics.l001.de
Methods
Imaging of Embryos Using Confocal Microscopy
• Confocal microscope detects fluorescence
• Embryos oriented to image dorsal region
• Anterior and posterior marked
• Imaged over a period of 4 hours
3D Image Reconstruction
Viewing Cell Divisions in Live
Embryos
Close Up: Cell Division
Viewing Cell Divisions with Spindle
Fibers Labeled
Tracking Cell Rotation During
Division: Before and After
3D Image Navigation
Analysis

Special computer program tracks division alignment


Point assigned to each half of
separating chromosomes
Program tracks and measures the
angle of division relative to the
anterior-posterior plane and dorsalventral plane
Image Courtesy of L. Caneparo
Results
We observed an active rotation of the cells to align
prior to division.
Before
After
Future Directions
• Further Analysis
• Quantify angle/character of rotation
• Statistically analyze data
• Investigate cell signaling patterns
• Compare cell division in other vertebrate models
• Explore implications on neural development
Acknowledgements
Special Thanks to:
• Luca Caneparo
• Fraser Lab
• Sherry Tsai
• James Maloney
• California Institute of Technology
• Howard Hughes Medical Institute
• Siemens Foundation
• Our families