The Effect of Erbb3 on Neural Crest Cells Christie Ojiaku Judith Eisen’s Lab

What is Erbb3? Receptor-type tyrosine kinase proto- oncogene Regulates cell processes and has roles in development Regulates cell processes and has roles in development Alterations may aid in onset of cancer or other abnormalities 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 TransparentCheap

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 Wouldn’t be able to survive in the wild without DRGs Could not sense pain No sensory stimuli Sense of movement would be disrupted 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 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? Does mutant lack DRGs early on, but go on to form as mutant ages? Does mutant lack DRGs early on, but go on to form as mutant ages? Complete lack of DRGs 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 HuC: DRGs, RBs Islet: DRGs, RBs Islet: DRGs, RBs Zn-8: DRGs, RBs Zn-8: DRGs, RBs Zn-12: RBs Zn-12: RBs Used antibodies as specific markers for DRGs and RBs Secondary antibody stained for fluorescence

Results – 1wpf HuCIslet WTWTWTWT MUTMUTMUTMUT

Zn 8 Zn 12 WTWTWTWT MUTMUTMUTMUT

Results – 2wpf HuCIslet WTWTWTWT MUTMUTMUTMUT

Zn 8 Zn 12 WTWTWTWT MUTMUTMUTMUT

Results – 4wpf HuCIslet WTWTWTWT MUTMUTMUTMUT

Zn 8 Zn 12 WTWTWTWT MUTMUTMUTMUT

Results – 6wpf HuCIslet WTWTWTWT MUTMUTMUTMUT

Zn 8 Zn 12 WTWTWTWT MUTMUTMUTMUT

Conclusion Erbb3 mutants lack DRGs Not present in early life Not present in early life Do not form later in mutants 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 Series of sensory pores along the side of the fish Series of sensory pores along the side of the fish Detects pressure changes and vibrations in water Detects pressure changes and vibrations in water Used for prey detection and predator avoidance 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

Acknowledgements GodSPUR Judith Eisen Yasuko Honjo Eisen Lab

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