Chlebowski, Mady

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Ithaca College

Madison	(Mady)	Chlebowski		Faculty	Advisor:	Ian	Woods			Abstract:	Somatosensory	neurons	enable	appropriate	responses	to	environmental	stimuli,	thereby	allowing	us	avoid	pain	and	danger	[1].	Different	populations	of	these	neurons	respond	to	various	types	of	sensory	stimuli	such	as	touch,	temperature,	chemical,	and	pain	[2].	Somatosensory	neurons	grow	by	branching	through	the	skin,	and	their	goal	is	to	maximize	coverage	[3].	Neuronal	migration	and	axonogenesis,	which	are	vital	to	this	proper	neuronal	development	require	the	activity	of	many	cellular	components	–	including	cytoplasmic	microtubules	[7].	Therefore,	proteins	in	charge	of	cytoskeletal	regulation	are	thought	to	help	control	and	guide	neuron	shape	[4,5].		Many	genes	are	involved	in	the	regulation	of	neuronal	structures	and	these	genes	have	the	potential	to	mediate	interactions	between	somatosensory	neurons	and	the	environment.	One	such	gene	family	is	the	tubulin	polymerization	promoting	proteins	(tppps)	which	contains	tppp,	tppp2,	and	tppp3	(figure	1).	Tppp	proteins	function	in	the	polymerization	of	the	microtubule	tubulin	in	vitro	[6].	Axonogenesis,	the	extension	of	a	single	immature	process	and	its	subsequent	differentiation	into	an	axon,	is	controlled	by	the	polarization	of	microtubules	[7].	Neuron	polarization	and	neurite	outgrowth	are	also	tightly	controlled	by	microtubule	stabilization	and	acetylation	[13],	and	altered	microtubule	stability	underlies	the	dynamic	nature	of	growth	and	retraction	processes	in	neurons	[7].	The	extensive	rearrangement	of	cell	shape	that	occurs	during	this	migration	is	mediated	by	changes	in	the	cytoskeleton	[8].	Previous	research	has	shown	that	microtubule	stabilization	is	sufficient	to	induce	axon	formation	and	that	moderate	microtubule	destabilization	selectively	reduces	the	formation	of	minor	neurites	[12].	Therefore,	tppp2	may	have	an	essential	role	in	regulating	the	shape	of	somatosensory	neurons	via	tubulin	stabilization.	While	the	biochemical	role	of	tppp	is	fairly	well	characterized	[6],	the	role	of	its	paralog	tppp2	is	relatively	unknown,	though,	tppp2	has	been	shown	to	be	expressed	in	sensory	neurons	[9].	Injection	of	antisense	morpholinos	targeting	zebrafish	tppp2	reduced	axon	extension	in	Rohon	Beard	neurons	(RB)	and	motorneurons,	suggesting	that	tubulin	polymerization	may	be	crucial	for	proper	axon	growth	[9].	Since	tppp2	functions	in	neuronal	stabilization,	I	hypothesize	that	a	knockout	of	the	tppp2	would	lead	to	decreased	stabilization	of	tubulin	and	thus	decrease	the	number	of	TGG	projections	formed.	My	research	goal	has	been	to	understand	the	role	of	tppp2	in	zebrafish	somatosensory	system	development	and	somatosensory	reception.	Previously	I	have	worked	to	identify	founders	for	CRISPR/Cas9-induced	mutations	of	tppp2,	performed	the	husbandry	to	cross		these	mutations	to	homozygosity.	I	then	have	prepared	assays	to	evaluate	and	analyze	loss	-of	function	mutations	focusing	on	development	of	neuronal	shape,	and	function	of	these	neurons	in	detecting	sensory	stimuli.			Via	confocal	imaging	of	acetylated	tubulin	antibody	tagged	neurons,	I	have	investigated	developmental	changes	to	trigeminal	ganglion	peripheral	projections,	a	population	of	sensory	neurons	in	the	head,	along	with	RB	neurons	in	the	tail	(figure	2).	I	hypothesize	that	there	will	be	a	decreased	number	of	peripheral	projections	in	my	KOs.	In	contrast	to	Aoki,	I	have	found	no	apparently	difference	between	KOs	and	their	wild-type	counterparts.	I	also	aim	to	elucidate	the	specific	function	of	these	projections	through	developmental	and	behavioral	assays	of	larval	zebrafish.	Preliminary	data	has	shown	that	these	tppp2	KO’s	may	respond	differently	to	chemical	stimuli	than	their	wild-type	or	heterozygous	counterparts,	but	respond	normally	to	thermal	stimuli	(figure	3).			As	described	above,	we	have	been	unable	to	replicate	the	findings	of	the	tppp2	MO	paper	in	our	knockouts	(KOs)	[9],	one	hypothesis	for	why	this	is	occurring	is	functional	compensation	due	to	gene	duplication.	This	would	mean	the	duplicated	versions	of	tppp2,	are	able	to	rescue	the	KO	[14].	Due	to	the	potential	functional	compensation	of	our	tppp2	knockouts	(KOs),	I	will	perform	experiments	to	determine	if	tppp2	overexpression	plays	a	role	in	the	dysregulation	of	peripheral	projection	development.			These	studies	will	provide	the	groundwork	for	determining	the	specific	mechanisms	underlying	the	role	of	microtubules	in	neuronal	development,	as	well	as	enhance	our	understanding	of	somatosensation.	Dysfunction	of	somatosensory	neurons	can	cause	chronic	pain	[11];	accordingly,	improved	understanding	of	sensory	neuron	development	and	function	could	enable	the	generation	of	treatments	for	chronic	pain	conditions.	Furthermore,	we	could	use	this	knowledge	to	enhance	our	understanding	of	diseases	causing	sensory	deficits	in	humans.		Figures:		Figure	1:	Phylogeny	of	tppp	protein	family	in	vertebrates.		Tppp	proteins	are	more	related	between	paralogs	than	within	species.							Figure	2:	Acetylated	Tubulin	antibody	staining	in	populations	of	developing	sensory	neurons	present	in	larval	zebrafish.	Rohon-Beard	Neurons	are	present	in	the	trunk	of	larval	zebrafish	and	Trigeminal	Ganglion	neurons	are	present	in	the	head.			Figure	3:	tppp2	Mutant	larval	zebrafish	show	decreased	response	to	chemical	(mustard	oil)	stimuli	compared	to	wildtype	and	heterozygous	fish.	Pixels	represent	difference	in	motion	over	time	analyzed,	total	pixels	represent	total	differences	in	movement.		Literature	cited:		[1]	Gau	P,	Poon	J,	Ufret-Vincenty	C,	Snelson	CD,	Gordon	SE,	Raible	DW,	Dhaka	A.	2013.	The	zebrafish	ortholog	of	TRPV1	is	required	for	heat-induced	locomotion.	J	Neurosci.	33(12):5249–5260.	doi:10.1523/JNEUROSCI.5403-12.2013.		[2]Lumpkin	EA,	Caterina	MJ.	2007.	Mechanisms	of	sensory	transduction	in	the	skin.	Nature.	445(7130):858–865.	doi:10.1038/nature05662.		[3]	Pan	YA,	Choy	M,	Prober	DA,	Schier	AF.	2012.	Robo2	determines	subtype-specific	axonal	projections	of	trigeminal	sensory	neurons.	Development.	139(3):591–600.	doi:10.1242/dev.076588.	[4]	Hunter,	Paul	R.,	Nikolas	Nikolaou,	Benjamin	Odermatt,	Philip	R.	Williams,	Uwe	Drescher,	and	Martin	P.	Meyer.	“Localization	of	Cadm2a	and	Cadm3	Proteins	during	Development	of	the	Zebrafish	Nervous	System.”	The	Journal	of	Comparative	Neurology	519,	no.	11	(August	1,	2011):	2252–70.	doi:10.1002/cne.22627		[5]	Kapitein,	Lukas	C.,	and	Casper	C.	Hoogenraad.	“Building	the	Neuronal	Microtubule		Cytoskeleton.”	Neuron	87,	no.	3	(August	2015):	492–506.	doi:10.1016/j.neuron.2015.05.046		[6]	Tirián	L,	Hlavanda	E,	Oláh	J,	Horváth	I,	Orosz	F,	Szabó	B,	Kovács	J,	Szabad	J,	Ovádi	J.	2003.	TPPP/p25	promotes	tubulin	assemblies	and	blocks	mitotic	spindle	formation.	PNAS.	100(24):13976–13981.	doi:10.1073/pnas.2436331100.		[7]	Sakakibara	A,	Ando	R,	Sapir	T,	Tanaka	T.	2013.	Microtubule	dynamics	in	neuronal	morphogenesis.	Open	Biol.	3(7).	doi:10.1098/rsob.130061.	[accessed	2019	Jan	4].	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728923/.		[8]	Feng	Y,	Walsh	CA.	2001.	Protein-protein	interactions,	cytoskeletal	regulation	and	neuronal	migration.	Nat	Rev	Neurosci.	2(6):408–416.	doi:10.1038/35077559.		[9]	Aoki	M,	Segawa	H,	Naito	M,	Okamoto	H.	2014.	Identification	of	possible	downstream	genes	required	for	the	extension	of	peripheral	axons	in	primary	sensory	neurons.	Biochem	Biophys	Res	Commun.	445(2):357–362.	doi:10.1016/j.bbrc.2014.01.193.		[10]	Palanca,	Ana	Marie	Silo.	2012.	“Somatosensory	Neuron	Subtypes	and	Sensory		Transduction	in	Larval	Zebrafish.”	EScholarship,	January.	http://escholarship.org/uc/item/2tg55242.		[11]	Patel,	Smruti	K.,	and	James	K.	Liu.	2016.	“Overview	and	History	of	Trigeminal		Neuralgia.”	Neurosurgery	Clinics	of	North	America	27	(3):265–76.	https://doi.org/10.1016/j.nec.2016.02.002.		[12]Witte	H,	Neukirchen	D,	Bradke	F.	2008.	Microtubule	stabilization	specifies	initial	neuronal	polarization.	J	Cell	Biol.	180(3):619–632.	doi:10.1083/jcb.200707042.		[13]	Perdiz	D,	Mackeh	R,	Poüs	C,	Baillet	A.	2011.	The	ins	and	outs	of	tubulin	acetylation:	More	than	just	a	post-translational	modification?	Cellular	Signalling.	23(5):763–771.	doi:10.1016/j.cellsig.2010.10.014.		[14]	Liang	H,	Li	W-H.	2009.	Functional	compensation	by	duplicated	genes	in	mouse.	Trends	Genet.	25(10):441–442.	doi:10.1016/j.tig.2009.08.001.		

The role of tppp2 in the development and function of somatosensory neurons in zebrafish

https://digitalcommons.ithaca.edu/cgi/viewcontent.cgi?article=1714&amp;context=whalen

The role of tppp2 in the development and function of somatosensory neurons in zebrafish

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