Uncx4.1 Function During Spinal Cord Development

Meg ChristDr. Michael Gross

HHMI 2005

Spinal Cord Injuries are Typically Abrupt and

Permanent

Car Falls Sports Violence/Other

40% 40%

12%

8%

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

Petri Dish

Embryonic Stem Cell

FGF

Sonic Hedgehog protein

Retinoic AcidMotor 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

affectother 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

Transcription of Normal Uncx4.1 Gene

Uncx4.1 Protein

5’ 3’

5’ 3’

Transcription of Knock-in Gene

Knock-in Gene

Uncx4.1 out

Effect

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

Add fluorescent Pax2 antibody that lights up all cells expressing Pax2.

Pax2 Knock-in

Gene

Add fluorescent antibodies to light up cells expressing the Knock-in gene.

Pax 2

There is currently no Uncx4.1 Antibody

Uncx4.1

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

Uncx4.1 Protein Binds to DNA to stop transcription of No Pax2 protein!

the Pax2 gene

Nonfunctional Protein Pax2 gene gets transcribed Pax2 Protein is expressed

Can’t bind!

One Interpretation of What Is Happening in a Yellow Cell

Nonfunctional Protein Pax2 gene gets transcribed Pax2 Protein is made

Can’t bind!

Initial Conclusion: One of the Functions of Uncx4.1 is to

Suppress Pax2

Repeat Antibody Staining Process for Many Different

Proteins to Understand Relationships

…you can start to assemble something like this.

Combine this with many other pathways…Nonfunctional Protein Pax2 gene gets

transcribed Pax2 Protein is expressed

Can’t bind!

After Pinpointing the Signaling Pathways for Interneuron Development, Replicate It

Petri Dish

Embryonic Stem Cell

Factors involved in interneuron formation

Interneurons

Thank You!!

The Howard Hughes Medical Institute

Michael

Kevin

Ben

Everyone else in the lab!