The Effect of The Effect of Erbb3Erbb3 on on Neural Crest CellsNeural Crest Cells

Christie OjiakuChristie Ojiaku

Judith Eisen’s LabJudith Eisen’s Lab

What is What is Erbb3Erbb3??

Receptor-type tyrosine kinase proto-Receptor-type tyrosine kinase proto-oncogeneoncogene Regulates cell processes and has roles in Regulates cell processes and has roles in

developmentdevelopment Alterations may aid in onset of cancer or other Alterations may aid in onset of cancer or other

abnormalitiesabnormalities

Mammals without Mammals without erbb3erbb3 do not exhibit do not exhibit proper neural crest cell migrationproper neural crest cell migration

What are neural crest cells?What are neural crest cells?

Located on neural tube’s outer surfaceLocated on neural tube’s outer surfaceMigrate ventrally from the neural tube after neurulation Migrate ventrally from the neural tube after neurulation Migration ends when neural crest cells accumulate at Migration ends when neural crest cells accumulate at barriers or after receiving a signal to cease migrationbarriers or after receiving a signal to cease migrationPluripotent; generate cartilage, connective tissue, Pluripotent; generate cartilage, connective tissue, pigment cells, dorsal root ganglia through migrationpigment cells, dorsal root ganglia through migration

What are dorsal root ganglia What are dorsal root ganglia (DRGs)?(DRGs)?

Swellings on the back of Swellings on the back of spinal nerves that contain spinal nerves that contain sensory neuronssensory neuronsPass sensory information Pass sensory information from skin, skeletal muscles, from skin, skeletal muscles, and joints to neurons in the and joints to neurons in the spinal cord which then spinal cord which then transmits the information to transmits the information to the brainthe brainFormed by migrating neural Formed by migrating neural crest cells pausing at the crest cells pausing at the appropriate spotsappropriate spotsLocated outside neural tubeLocated outside neural tube

Using Zebrafish as a ModelUsing Zebrafish as a Model

Development similar Development similar to that of human to that of human embryoembryo

Fast rate of Fast rate of developmentdevelopment

Develop outside Develop outside mother’s bodymother’s body

TransparentTransparent

CheapCheap

PurposePurposeErbb3Erbb3 mutant zebrafish show some mutant zebrafish show some traits of improper neural crest cell traits of improper neural crest cell migrationmigrationShouldn’t these fish also lack DRGs? Shouldn’t these fish also lack DRGs?

Wouldn’t be able to survive in Wouldn’t be able to survive in the wild without DRGsthe wild without DRGs

Could not sense painCould not sense painNo sensory stimuliNo sensory stimuli

Sense of movement would be Sense of movement would be disrupteddisrupted

In humans, DRGs play a In humans, DRGs play a role in helping us know the role in helping us know the position of limbs relative to position of limbs relative to bodybodyEx: watching TV and Ex: watching TV and snackingsnackingFish lacking DRGs should Fish lacking DRGs should not be able to swim not be able to swim properlyproperly

Seems impossible that mutants Seems impossible that mutants could lack DRGs and still could lack DRGs and still function without helpfunction without help

Rohon-Beard neurons (RBs)Rohon-Beard neurons (RBs)

Embryonic sensory Embryonic sensory neuronsneurons

Present in fish until Present in fish until around 1wpfaround 1wpf

Found inside neural Found inside neural tubetube

Experimental QuestionsExperimental Questions

Do Do erbb3erbb3 mutants have DRGs? mutants have DRGs? Does mutant lack DRGs early on, but go on to form Does mutant lack DRGs early on, but go on to form

as mutant ages?as mutant ages? Complete lack of DRGsComplete lack of DRGs

If DRGs never appear, do mutants retain RBs?If DRGs never appear, do mutants retain RBs?

Finding the GenotypeFinding the Genotype

Crossed and grew fish until 1wpf, 2wpf, 4wpf, and 6wpfCrossed and grew fish until 1wpf, 2wpf, 4wpf, and 6wpf

Extracted DNA from heads for use in genotyping the fish Extracted DNA from heads for use in genotyping the fish

Amplified DNA using PCRAmplified DNA using PCR

Fixed remainder of body to preserve it for mounting on Fixed remainder of body to preserve it for mounting on slidesslides

Finding the GenotypeFinding the Genotype

Hpy8I cut DNA sequence present in WT fish Hpy8I cut DNA sequence present in WT fish WT DNA cut with Hpy8I was shorter and ran WT DNA cut with Hpy8I was shorter and ran fasterfasterRan DNA gel to determine which fish were Ran DNA gel to determine which fish were mutant, heterozygous mutant, or WTmutant, heterozygous mutant, or WT

MountingMounting

Mounted mutant and Mounted mutant and WT fish and had them WT fish and had them cut cross-sectionally cut cross-sectionally from neck to anus from neck to anus and placed on slidesand placed on slides

StainingStaining

Stained all slides with four primary antibodiesStained all slides with four primary antibodies HuC: DRGs, RBsHuC: DRGs, RBs Islet: DRGs, RBsIslet: DRGs, RBs Zn-8: DRGs, RBs Zn-8: DRGs, RBs Zn-12: RBsZn-12: RBs

Used antibodies as specific markers for DRGs and RBsUsed antibodies as specific markers for DRGs and RBsSecondary antibody stained for fluorescenceSecondary antibody stained for fluorescence

Results – 1wpfResults – 1wpfHuCHuC IsletIslet

W W T T

MMUU TT

Results – 1wpfResults – 1wpfZn 8 Zn 8 Zn 12Zn 12

WWTT

MMUUTT

Results – 2wpfResults – 2wpfHuCHuC IsletIslet

WWTT

MMUUTT

Results – 2wpfResults – 2wpfZn 8Zn 8 Zn 12Zn 12

WWTT

MMUUTT

Results – 4wpfResults – 4wpfHuCHuC IsletIslet

WWTT

MMUUTT

Results – 4wpfResults – 4wpfZn 8Zn 8 Zn 12Zn 12

WWTT

MMUUTT

Results – 6wpfResults – 6wpfHuCHuC IsletIslet

WWTT

MMUUTT

Results – 6wpfResults – 6wpfZn 8Zn 8 Zn 12Zn 12

WWTT

MMUUTT

ConclusionConclusion

Erbb3Erbb3 mutants lack DRGs mutants lack DRGs Not present in early lifeNot present in early life Do not form later in mutantsDo not form later in mutants

Erbb3Erbb3 mutants initially have RBs, but lose mutants initially have RBs, but lose them at the same time as WT fishthem at the same time as WT fish

Some other way mutants are able to Some other way mutants are able to sense without DRGs or RBssense without DRGs or RBs

ConclusionConclusion

Lateral LineLateral Line Series of sensory pores along the side of the fishSeries of sensory pores along the side of the fish Detects pressure changes and vibrations in waterDetects pressure changes and vibrations in water Used for prey detection and predator avoidanceUsed for prey detection and predator avoidance

ConclusionConclusion

Explains how fish could feel sensory Explains how fish could feel sensory stimuli but not how fish are able to stimuli but not how fish are able to coordinate their movementscoordinate their movements

Further studies need to be conductedFurther studies need to be conducted

ReferencesReferenceshttp://www.brown.edu/Courses/BI0032/neurcrst/ncori.htmhttp://www.nature.com/hdy/journal/v97/n3/full/6800867a.htmlhttp://www.trinity.edu/rblyston/craftyMol/ncm.htmhttp://www.trinity.edu/rblyston/craftyMol/ncm.htmhttp://www.wdv.com/Cancer/Pain/Anatomy/main.htmlhttp://www.wdv.com/Cancer/Pain/Anatomy/main.htmlhttp://www.answers.com/topic/neural-cresthttp://www.answers.com/topic/neural-cresthttp://www.le.ac.uk/biology/staff/jm/jmres.htmhttp://www.le.ac.uk/biology/staff/jm/jmres.htmhttp://hopes.stanford.edu/rltdsci/trinuc/f3.htmlhttp://hopes.stanford.edu/rltdsci/trinuc/f3.htmlhttp://depts.washington.edu/uweek/archives/1998.02.FEB_12/article9.htmlhttp://depts.washington.edu/uweek/archives/1998.02.FEB_12/article9.htmlhttp://neuroscience.uth.tmc.edu/s2/ii3-3.htmlhttp://neuroscience.uth.tmc.edu/s2/ii3-3.html

AcknowledgementsAcknowledgements

GodGod

SPURSPUR

Judith EisenJudith Eisen

Yasuko HonjoYasuko Honjo

Eisen LabEisen Lab

THE END!THE END!