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

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
Effect
5’
Transcription of Normal Uncx4.1 Gene
3’
Uncx4.1 Protein
5’
Transcription of Knock-in Gene
Uncx4.1 out
3’
Knock-in Gene
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
Add fluorescent antibodies to light up cells expressing the Knock-in gene.
Uncx4.1
Pax 2
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 Pax2 gene gets transcribed Pax2 Protein is expressed
Uncx4.1 Protein Binds to DNA to stop transcription of the Pax2 gene
No Pax2 protein!

One Interpretation of What Is Happening in a Yellow Cell
Nonfunctional Protein
Can’t bind!
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!
Combine this with many other pathways…
Nonfunctional Protein
Pax2 Protein is expressed
Pax2 gene gets transcribed
…you can start to assemble something like this.

After Pinpointing the Signaling Pathways for Interneuron Development, Replicate It
Embryonic Stem Cell
Factors involved in interneuron formation
Petri Dish Interneurons

The Howard Hughes Medical Institute
Michael
Kevin
Ben
Everyone else in the lab!