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Uncx4.1 Function During Spinal Cord
Development
Meg Christ
Dr. Michael Gross
HHMI 2005
Spinal Cord Injuries are Typically Abrupt
and Permanent
40%
40%
12%
8%
Car
Falls
Sports
Violence/Other
What Happens in a Spinal Cord Injury
Bone pokes
Severs Interneurons
Neurons Run the Length of the Spinal
Cord and Do Not Naturally Repair
Themselves
Interneurons Connect Your Brain and Body
Porcupine example: even though they’re both in
your arm, can’t talk to each other. Must go
through brain via interneurons.
If Interneurons Are Severed, How Can They
Be Repaired?
Jessell Studies the Development of Motor
Neurons at the Cellular Level
In order to guide stem cell differentiation, must understand body’s natural mechanism of differentiation and
in order to do that you have to study development so here’s the 3 pictures of development and the molecule
it needs to develop that way (FGF, shh, retinoic).
Jessell Then Replicates What the Body Does
Naturally In a Petri Dish
Embryonic Stem Cell
FGF
Sonic Hedgehog protein
Retinoic Acid
Petri Dish
Motor
neurons
Paralyzed Patients Regain Some Lost Motor
Function Upon Injection of Motor Neurons
Motor
neurons
Some motor function
Motor neurons atrophy when interneurons are broken.
The next step is restoring the ability to sense and then act on something,
and that requires interneurons.
Follow A Similar Procedure: Start by
Looking at Development
Neural Tube
(remember to
mention that each
of these
corresponds to a
particular
population of
interneurons)
Uncx4.1 Is a Gene Expressed In Some of
These Populations
First Question:
Which ones?
Second Question:
How does Uncx4.1 affect
other genes’ expression?
How Do We Know Uncx4.1 Is Needed In
The First Place?
Do they
need me?
To find out if it is important, make it non-functional and see what effect it has.
Previous Research Has Created a Knock-in
Gene
5’
Effect
3’
Uncx4.1 Protein
Transcription of Normal Uncx4.1 Gene
Uncx4.1 out
5’
3’
Transcription of Knock-in Gene
Knock-in Gene
Effect
Phenotypic Effect
But we’d like to know what’s going on molecularly, and with respect to all the
other genes expressed in the neural tube!
Cross Mice to Yield Uncx4.1 Wildtype,
Heterozygote and Mutant Embryos
Wildtype
Heterozygote
Mutant
Comparing Wildtypes and Mutants Section
by Section
Wildtype
Mutant
Label Specific Cells With Fluorescent
Antibody Markers to Answer Both
Questions Posed
Pax2
Knock-in
Gene
Uncx4.1
Pax 2
Add fluorescent antibodies to light up cells expressing
the Knock-in gene.
There is currently no Uncx4.1 Antibody
Add fluorescent Pax2 antibody that lights up all cells
expressing Pax2.
Answering the First Question: Which
Populations is Uncx4.1 Expressed In?
(there’s a stain here, with just the
green channel on)
Yellow Indicates Both Pax2 and Knock-In
Gene Expression
Compare Pax2 Expression of This Mutant
With That of a Wildtype
Wildtype and mutant, red
channel only
One Possible Interpretation for the Pax2
and Uncx4.1 Relationship
Can’t bind!
Nonfunctional Protein
Uncx4.1 Protein
Pax2 gene gets transcribed
Binds to DNA
to stop transcription of
the Pax2 gene
Pax2 Protein is expressed
No Pax2 protein!
One Interpretation of What Is Happening in
a Yellow Cell
Can’t bind!
Nonfunctional Protein
Pax2 gene gets transcribed
Pax2 Protein is made
Initial Conclusion: One of the Functions of
Uncx4.1 is to Suppress Pax2
Repeat Antibody Staining Process for Many
Different Proteins to Understand
Relationships
Can’t bind!
Nonfunctional Protein
Pax2 gene gets transcribed
Pax2 Protein is expressed
…you can start to
assemble something
like this.
Combine this with many
other pathways…
After Pinpointing the Signaling Pathways
for Interneuron Development, Replicate It
Embryonic Stem Cell
Factors involved in
interneuron formation
Petri Dish
Interneurons
Thank You!!
The Howard Hughes Medical Institute
Michael
Kevin
Ben
Everyone else in the lab!