Transcript The Effect of Erbb3 on Neural Crest Cells

The Effect of Erbb3 on
Neural Crest Cells
Christie Ojiaku
Judith Eisen’s Lab
What is Erbb3?
Receptor-type tyrosine kinase protooncogene


Regulates cell processes and has roles in
development
Alterations may aid in onset of cancer or other
abnormalities
Mammals without erbb3 do not exhibit
proper neural crest cell migration
What are neural crest cells?
Located on neural tube’s outer surface
Migrate ventrally from the neural tube after neurulation
Migration ends when neural crest cells accumulate at
barriers or after receiving a signal to cease migration
Pluripotent; generate cartilage, connective tissue,
pigment cells, dorsal root ganglia through migration
What are dorsal root ganglia
(DRGs)?
Swellings on the back of
spinal nerves that contain
sensory neurons
Pass sensory information
from skin, skeletal muscles,
and joints to neurons in the
spinal cord which then
transmits the information to
the brain
Formed by migrating neural
crest cells pausing at the
appropriate spots
Located outside neural tube
Using Zebrafish as a Model
Development similar
to that of human
embryo
Fast rate of
development
Develop outside
mother’s body
Transparent
Cheap
Purpose
Erbb3 mutant zebrafish show some
traits of improper neural crest cell
migration
Shouldn’t these fish also lack DRGs?

Wouldn’t be able to survive in
the wild without DRGs
Could not sense pain
No sensory stimuli

Sense of movement would be
disrupted
In humans, DRGs play a
role in helping us know the
position of limbs relative to
body
Ex: watching TV and
snacking
Fish lacking DRGs should
not be able to swim
properly

Seems impossible that mutants
could lack DRGs and still
function without help
Rohon-Beard neurons (RBs)
Embryonic sensory
neurons
Present in fish until
around 1wpf
Found inside neural
tube
Experimental Questions
Do erbb3 mutants have DRGs?
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
Does mutant lack DRGs early on, but go on to form
as mutant ages?
Complete lack of DRGs
If DRGs never appear, do mutants retain RBs?
Finding the Genotype
Crossed and grew fish until 1wpf, 2wpf,
4wpf, and 6wpf
Extracted DNA from heads for use in
genotyping the fish
Amplified DNA using PCR
Fixed remainder of body to preserve it for
mounting on slides
Finding the Genotype
Hpy8I cut DNA sequence present in WT fish
WT DNA cut with Hpy8I was shorter and ran
faster
Ran DNA gel to determine which fish were
mutant, heterozygous mutant, or WT
Mounting
Mounted mutant and
WT fish and had them
cut cross-sectionally
from neck to anus
and placed on slides
Staining
Stained all slides with four primary antibodies
 HuC: DRGs, RBs
 Islet: DRGs, RBs
 Zn-8: DRGs, RBs
 Zn-12: RBs
Used antibodies as specific markers for
DRGs and RBs
Secondary antibody stained for fluorescence
Results – 1wpf
HuC
W
T
M
U
T
Islet
Results – 1wpf
Zn 8
W
T
M
U
T
Zn 12
Results – 2wpf
HuC
W
T
M
U
T
Islet
Results – 2wpf
Zn 8
W
T
M
U
T
Zn 12
Results – 4wpf
HuC
W
T
M
U
T
Islet
Results – 4wpf
Zn 8
W
T
M
U
T
Zn 12
Results – 6wpf
HuC
W
T
M
U
T
Islet
Results – 6wpf
Zn 8
W
T
M
U
T
Zn 12
Conclusion
Erbb3 mutants lack DRGs
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Not present in early life
Do not form later in mutants
Erbb3 mutants initially have RBs, but lose
them at the same time as WT fish
Some other way mutants are able to
sense without DRGs or RBs
Conclusion
Lateral Line
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Series of sensory pores along the side of the fish
Detects pressure changes and vibrations in water
Used for prey detection and predator avoidance
Conclusion
Explains how fish could feel sensory
stimuli but not how fish are able to
coordinate their movements
Further studies need to be conducted
References
http://www.brown.edu/Courses/BI0032/neurcrst/ncori.htm
http://www.nature.com/hdy/journal/v97/n3/full/6800867a.html
http://www.trinity.edu/rblyston/craftyMol/ncm.htm
http://www.wdv.com/Cancer/Pain/Anatomy/main.html
http://www.answers.com/topic/neural-crest
http://www.le.ac.uk/biology/staff/jm/jmres.htm
http://hopes.stanford.edu/rltdsci/trinuc/f3.html
http://depts.washington.edu/uweek/archives/1998.02.FEB_12/article9.html
http://neuroscience.uth.tmc.edu/s2/ii3-3.html
Acknowledgements
God
SPUR
Judith Eisen
Yasuko Honjo
Eisen Lab
THE END!