Uncx4.1 Function During Spinal Cord Development

1. Uncx4.1 Function During Spinal Cord Development Meg Christ Dr. Michael Gross HHMI 2005

2. Spinal Cord Injuries are Typically Abrupt and Permanent 40% 40% 12% 8% Car Falls Sports Violence/Other

3. What Happens in a Spinal Cord Injury Bone pokes Severs Interneurons

4. Neurons Run the Length of the Spinal Cord and Do Not Naturally Repair Themselves

5. 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.

6. If Interneurons Are Severed, How Can They Be Repaired?

7. 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).

8. Embryonic Stem Cell FGF Sonic Hedgehog protein Retinoic Acid Petri Dish Motor neurons Jessell Then Replicates What the Body Does Naturally In a Petri Dish

9. Motor neurons Paralyzed Patients Regain Some Lost Motor Function Upon Injection of 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.

10. 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)

11. 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?

12. 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.

13. Previous Research Has Created a Knock-in Gene Effect 5’ 3’ Uncx4.1 Protein Transcription of Normal Uncx4.1 Gene Uncx4.1 out Effect 5’ 3’ Transcription of Knock-in Gene Knock-in Gene

14. 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!

15. Cross Mice to Yield Uncx4.1 Wildtype, Heterozygote and Mutant Embryos Wildtype Heterozygote Mutant

16. Comparing Wildtypes and Mutants Section by Section Wildtype Mutant

17. Label Specific Cells With Fluorescent Antibody Markers to Answer Both Questions Posed Add fluorescent antibodies to light up cells expressing the Knock-in gene. Pax2 Knock-in Gene There is currently no Uncx4.1 Antibody Uncx4.1 Add fluorescent Pax2 antibody that lights up all cells expressing Pax2. Pax 2

18. Answering the First Question: Which Populations is Uncx4.1 Expressed In? (there’s a stain here, with just the green channel on)

19. Yellow Indicates Both Pax2 and Knock-In Gene Expression

20. Compare Pax2 Expression of This Mutant With That of a Wildtype Wildtype and mutant, red channel only

21. Uncx4.1 Protein Binds to DNA to stop transcription of No Pax2 protein! the Pax2 gene Can’t bind! Nonfunctional Protein Pax2 gene gets transcribed Pax2 Protein is expressed One Possible Interpretation for the Pax2 and Uncx4.1 Relationship

22. One Interpretation of What Is Happening in a Yellow Cell Can’t bind! Nonfunctional Protein Pax2 gene gets transcribed Pax2 Protein is made

23. Initial Conclusion: One of the Functions of Uncx4.1 is to Suppress Pax2

26. Repeat Antibody Staining Process for Many Different Proteins to Understand Relationships Can’t bind! Combine this with many other pathways… Nonfunctional Protein Pax2 gene gets transcribed Pax2 Protein is expressed …you can start to assemble something like this.

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

28. Thank You!! The Howard Hughes Medical Institute Michael Kevin Ben Everyone else in the lab!