Gli3 is a Hedgehog (Hh) responsive transcription factor that can function as a transcriptional repressor or activator. We show that Gli3 activity in thymic epithelial cells (TEC) promotes positive selection and differentiation from CD4+CD8+ to CD4+CD8- single positive (SP4) cell in the fetal thymus and that Gli3 represses Shh Constitutive deletion of Gli3, and conditional deletion of Gli3 from TEC, reduced differentiation to SP4, whereas conditional deletion of Gli3 from thymocytes did not. Conditional deletion of Shh from TEC increased differentiation to SP4, and expression of Shh was upregulated in the Gli3-deficient thymus. Use of a transgenic Hh-reporter showed that the Hh pathway was active in thymocytes, and increased in the Gli3-deficient fetal thymus. Neutralisation of endogenous Hh proteins in the Gli3-/- thymus restored SP4 differentiation, indicating that Gli3 in TEC promotes SP4 differentiation by repression of Shh Transcriptome analysis showed that Hh-mediated transcription was increased but TCR-mediated transcription decreased in Gli3-/- thymocytes compared to WT

Crompton, T

Lau, C-I

Li, J

Papaioannou, E

Ross, S

Saldaña, JI

Solanki, A

Yanez, DC

UCL Discovery

S1Gli3+/+ Gli3-/- Gli3+/+ Gli3-/-ABCountDevelopment 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary informationFigure S1: Expression of TCRβ and TCRγδ in WT and Gli3-deficient E17.5+4 Days FTOCs. (A) Histograms showing the expression of TCRβ, gated on SP4 cells from Gli3+/+ and Gli3-/-. (B) Scatter plot showing the percentage of TCRβ+ and TCRγδ+ cells in the SP4 population from Gli3+/+ and Gli3-/- (n=3). Development 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary informationS2ABCDEFPC1 PC2 PC3 PC4 PC5PC1 PC2 PC3 PC4 PC5PC1 PC2 PC3 PC4 PC528%22%19%17%14%31%20%18%16%15%32%19%18%16%15%EigenvalueEigenvalueEigenvalueDevelopment 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary informationFigure S2: Principal Component Analyses of E18.5 WT and Gli3-mutant facs-sorted CD69-DP, CD69+DP and SP4 populations. PCA of the CD69-DP (A), the CD69+DP (C) and SP4 (E) datasets. Eigenvalues and percentage variability associated with each principal component for CD69-DP (B), the CD69+DP (D) and SP4 (F). Development 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary informationTable S1: Summary of intersected DEG and PCA genes annotated in the heatmap (Figure 6) of known relevant function. Gene Name Function in Thymus Reference TCR repertoire selection GenesEgr1 Egr1 is rapidly upregulated after TCR stimulation and increased Egr1 expression increases thymic selection. (Shao et al., 1997) Egr2 Egr2-deficiency impairs positive selection of both CD4 and CD8 SP thymocytes. It also upregulates survival molecule Bcl-2 during positive selection allowing survival of positively selected thymocytes (Lauritsen et al., 2008) Nab1 The Nab family members interact with the Egr1/2 to regulate different function in thymocytes and T cells. (Collins et al., 2008; Decker et al., 2003) Nab2 Itk Itk is very importance for the efficient positive and negative selection of thymocytes. (Andreotti et al., 2010) Tox TOX is important for the positive selection towards the CD4+ T cell lineage in the thymus (Aliahmad et al., 2011) Lef1 LEF-1 is important for the positive selection of CD4 SP thymocytes from the DP stage. Lef1 directly induce the master regulator of CD4 lineage commitment, ThPok. (Steinke et al., 2014) Pten Loss of Pten leads to defects in negative selection  (Suzuki et al., 2001) Socs1 Socs1 deficiency leads to impaired positive and negative selection in the thymus. Socs1 is important for CD4 T cell development. (Catlett and Hedrick, 2005) Id2 Id2 and Id3 allow CD8+ T cell development (Jones-Mason et al., 2012) Fas Fas-FasL interaction mediates activation-induced cell death of mature postthymic T cells, allowing effective negative selection and elimination of autoimmune cells (Kishimoto and Sprent, 1997) CamK4 CaMK4 regulates the Ca2+-dependent gene transcription, and loss of CaMK4 in thymocytes impairs positive selection. (Raman et al., 2001) Cd6 Increase in CD6 expression allow DP thymocytes differentiate to a SPs.  (Singer et al., 2002) Development 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary informationCd53 CD53 expression is correlated with positive selection. DP thymocytes expressing CD53 are undergoing selection or have just undergone selection. (Puls et al., 2002)  TCR signal strength modulators Cd81 Loss of CD81 in thymocytes increases the TCR signal strength (Cevik et al., 2012) Cd5 High cell surface CD5 expression correlates with a stronger TCR signal and vice versa   (Azzam et al., 2001) Nr4a1  The expression of the Nr4a family member is also correlated with increases in TCR signal strength. (Moran et al., 2011; Nowyhed et al., 2015) Nr4a3  Hedgehog Signalling and Target Genes Ihh Hedgehog protein expressed in DP cells  (Outram et al., 2009) Bmp2 Hedgehog signalling target involved in T-cell development in thymus  (Hager-Theodorides et al., 2002; Outram et al., 2000) Hoxa7 Homeobox (Hox) family members are targets of Hedgehog signalling and interact with various Hh family members to control developmental processes. (Roberts et al., 1995) H19 Hh signaling increases H19 expression (Chan et al., 2014) Kif13b Kif13b promotes Shh signalling (Schou et al., 2017) Tgfb1 TGF-β-increases Shh signalling to induced fibrosis.  (Chung and Fu, 2013) Itgav Itgav is a target of Hedgehog signalling and can modulate Hedgehog signalling (Kosinski et al., 2010; Zhou et al., 2013) Aliahmad,	P.,	Kadavallore,	A.,	de	la	Torre,	B.,	Kappes,	D.,	Kaye,	J.,	2011.	TOX	is	required	for	development	of	the	CD4	T	cell	lineage	gene	program.	J	Immunol	187,	5931-5940.	Andreotti,	A.H.,	Schwartzberg,	P.L.,	Joseph,	R.E.,	Berg,	L.J.,	2010.	T-cell	signaling	regulated	by	the	Tec	family	kinase,	Itk.	Cold	Spring	Harb	Perspect	Biol	2,	a002287.	Development 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary informationAzzam,	H.S.,	DeJarnette,	J.B.,	Huang,	K.,	Emmons,	R.,	Park,	C.S.,	Sommers,	C.L.,	El-Khoury,	D.,	Shores,	E.W.,	Love,	P.E.,	2001.	Fine	tuning	of	TCR	signaling	by	CD5.	J	Immunol	166,	5464-5472.	Catlett,	I.M.,	Hedrick,	S.M.,	2005.	Suppressor	of	cytokine	signaling	1	is	required	for	the	differentiation	of	CD4+	T	cells.	Nat	Immunol	6,	715-721.	Cevik,	S.I.,	Keskin,	N.,	Belkaya,	S.,	Ozlu,	M.I.,	Deniz,	E.,	Tazebay,	U.H.,	Erman,	B.,	2012.	CD81	interacts	with	the	T	cell	receptor	to	suppress	signaling.	PLoS	One	7,	e50396.	Chan,	L.H.,	Wang,	W.,	Yeung,	W.,	Deng,	Y.,	Yuan,	P.,	Mak,	K.K.,	2014.	Hedgehog	signaling	induces	osteosarcoma	development	through	Yap1	and	H19	overexpression.	Oncogene	33,	4857-4866.	Chung,	Y.,	Fu,	E.,	2013.	Crosstalk	between	Shh	and	TGF-beta	signaling	in	cyclosporine-enhanced	cell	proliferation	in	human	gingival	fibroblasts.	PLoS	One	8,	e70128.	Collins,	S.,	Lutz,	M.A.,	Zarek,	P.E.,	Anders,	R.A.,	Kersh,	G.J.,	Powell,	J.D.,	2008.	Opposing	regulation	of	T	cell	function	by	Egr-1/NAB2	and	Egr-2/Egr-3.	Eur	J	Immunol	38,	528-536.	Decker,	E.L.,	Nehmann,	N.,	Kampen,	E.,	Eibel,	H.,	Zipfel,	P.F.,	Skerka,	C.,	2003.	Early	growth	response	proteins	(EGR)	and	nuclear	factors	of	activated	T	cells	(NFAT)	form	heterodimers	and	regulate	proinflammatory	cytokine	gene	expression.	Nucleic	Acids	Res	31,	911-921.	Hager-Theodorides,	A.L.,	Outram,	S.V.,	Shah,	D.K.,	Sacedon,	R.,	Shrimpton,	R.E.,	Vicente,	A.,	Varas,	A.,	Crompton,	T.,	2002.	Bone	morphogenetic	protein	2/4	signaling	regulates	early	thymocyte	differentiation.	J	Immunol	169,	5496-5504.	Jones-Mason,	M.E.,	Zhao,	X.,	Kappes,	D.,	Lasorella,	A.,	Iavarone,	A.,	Zhuang,	Y.,	2012.	E	protein	transcription	factors	are	required	for	the	development	of	CD4(+)	lineage	T	cells.	Immunity	36,	348-361.	Kishimoto,	H.,	Sprent,	J.,	1997.	Negative	selection	in	the	thymus	includes	semimature	T	cells.	J	Exp	Med	185,	263-271.	Kosinski,	C.,	Stange,	D.E.,	Xu,	C.,	Chan,	A.S.,	Ho,	C.,	Yuen,	S.T.,	Mifflin,	R.C.,	Powell,	D.W.,	Clevers,	H.,	Leung,	S.Y.,	Chen,	X.,	2010.	Indian	hedgehog	regulates	intestinal	stem	cell	fate	through	epithelial-mesenchymal	interactions	during	development.	Gastroenterology	139,	893-903.	Lauritsen,	J.P.,	Kurella,	S.,	Lee,	S.Y.,	Lefebvre,	J.M.,	Rhodes,	M.,	Alberola-Ila,	J.,	Wiest,	D.L.,	2008.	Egr2	is	required	for	Bcl-2	induction	during	positive	selection.	J	Immunol	181,	7778-7785.	Moran,	A.E.,	Holzapfel,	K.L.,	Xing,	Y.,	Cunningham,	N.R.,	Maltzman,	J.S.,	Punt,	J.,	Hogquist,	K.A.,	2011.	T	cell	receptor	signal	strength	in	Treg	and	iNKT	cell	development	demonstrated	by	a	novel	fluorescent	reporter	mouse.	J	Exp	Med	208,	1279-1289.	Nowyhed,	H.N.,	Huynh,	T.R.,	Blatchley,	A.,	Wu,	R.,	Thomas,	G.D.,	Hedrick,	C.C.,	2015.	The	nuclear	receptor	nr4a1	controls	CD8	T	cell	development	through	transcriptional	suppression	of	runx3.	Sci	Rep	5,	9059.	Outram,	S.V.,	Hager-Theodorides,	A.L.,	Shah,	D.K.,	Rowbotham,	N.J.,	Drakopoulou,	E.,	Ross,	S.E.,	Lanske,	B.,	Dessens,	J.T.,	Crompton,	T.,	2009.	Indian	hedgehog	(Ihh)	both	promotes	and	restricts	thymocyte	differentiation.	Blood	113,	2217-2228.	Outram,	S.V.,	Varas,	A.,	Pepicelli,	C.V.,	Crompton,	T.,	2000.	Hedgehog	signaling	regulates	differentiation	from	double-negative	to	double-positive	thymocyte.	Immunity	13,	187-197.	Development 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary informationPuls,	K.L.,	Hogquist,	K.A.,	Reilly,	N.,	Wright,	M.D.,	2002.	CD53,	a	thymocyte	selection	marker	whose	induction	requires	a	lower	affinity	TCR-MHC	interaction	than	CD69,	but	is	up-regulated	with	slower	kinetics.	Int	Immunol	14,	249-258.	Raman,	V.,	Blaeser,	F.,	Ho,	N.,	Engle,	D.L.,	Williams,	C.B.,	Chatila,	T.A.,	2001.	Requirement	for	Ca2+/calmodulin-dependent	kinase	type	IV/Gr	in	setting	the	thymocyte	selection	threshold.	J	Immunol	167,	6270-6278.	Roberts,	D.J.,	Johnson,	R.L.,	Burke,	A.C.,	Nelson,	C.E.,	Morgan,	B.A.,	Tabin,	C.,	1995.	Sonic	hedgehog	is	an	endodermal	signal	inducing	Bmp-4	and	Hox	genes	during	induction	and	regionalization	of	the	chick	hindgut.	Development	121,	3163-3174.	Schou,	K.B.,	Mogensen,	J.B.,	Morthorst,	S.K.,	Nielsen,	B.S.,	Aleliunaite,	A.,	Serra-Marques,	A.,	Furstenberg,	N.,	Saunier,	S.,	Bizet,	A.A.,	Veland,	I.R.,	Akhmanova,	A.,	Christensen,	S.T.,	Pedersen,	L.B.,	2017.	KIF13B	establishes	a	CAV1-enriched	microdomain	at	the	ciliary	transition	zone	to	promote	Sonic	hedgehog	signalling.	Nat	Commun	8,	14177.	Shao,	H.,	Kono,	D.H.,	Chen,	L.Y.,	Rubin,	E.M.,	Kaye,	J.,	1997.	Induction	of	the	early	growth	response	(Egr)	family	of	transcription	factors	during	thymic	selection.	J	Exp	Med	185,	731-744.	Singer,	N.G.,	Fox,	D.A.,	Haqqi,	T.M.,	Beretta,	L.,	Endres,	J.S.,	Prohaska,	S.,	Parnes,	J.R.,	Bromberg,	J.,	Sramkoski,	R.M.,	2002.	CD6:	expression	during	development,	apoptosis	and	selection	of	human	and	mouse	thymocytes.	Int	Immunol	14,	585-597.	Steinke,	F.C.,	Yu,	S.,	Zhou,	X.,	He,	B.,	Yang,	W.,	Zhou,	B.,	Kawamoto,	H.,	Zhu,	J.,	Tan,	K.,	Xue,	H.H.,	2014.	TCF-1	and	LEF-1	act	upstream	of	Th-POK	to	promote	the	CD4(+)	T	cell	fate	and	interact	with	Runx3	to	silence	Cd4	in	CD8(+)	T	cells.	Nat	Immunol	15,	646-656.	Suzuki,	A.,	Yamaguchi,	M.T.,	Ohteki,	T.,	Sasaki,	T.,	Kaisho,	T.,	Kimura,	Y.,	Yoshida,	R.,	Wakeham,	A.,	Higuchi,	T.,	Fukumoto,	M.,	Tsubata,	T.,	Ohashi,	P.S.,	Koyasu,	S.,	Penninger,	J.M.,	Nakano,	T.,	Mak,	T.W.,	2001.	T	cell-specific	loss	of	Pten	leads	to	defects	in	central	and	peripheral	tolerance.	Immunity	14,	523-534.	Zhou,	X.,	Qiu,	W.,	Sathirapongsasuti,	J.F.,	Cho,	M.H.,	Mancini,	J.D.,	Lao,	T.,	Thibault,	D.M.,	Litonjua,	A.A.,	Bakke,	P.S.,	Gulsvik,	A.,	Lomas,	D.A.,	Beaty,	T.H.,	Hersh,	C.P.,	Anderson,	C.,	Geigenmuller,	U.,	Raby,	B.A.,	Rennard,	S.I.,	Perrella,	M.A.,	Choi,	A.M.,	Quackenbush,	J.,	Silverman,	E.K.,	2013.	Gene	expression	analysis	uncovers	novel	hedgehog	interacting	protein	(HHIP)	effects	in	human	bronchial	epithelial	cells.	Genomics	101,	263-272.	Table S2: PCA Gene list with relevant PC scores and Intersected Gene list for each sorted thymocyte population.Click here to Download Table S2Development 145: doi:10.1242/dev.146910: Supplementary informationDevelopment • Supplementary information

In the fetal thymus, Gli3 in thymic epithelial cells promotes thymocyte positive selection and differentiation by repression of Shh

http://discovery.ucl.ac.uk/10042367/7/Solanki_In_fetal_thymus_Suppl.pdf

In the fetal thymus, Gli3 in thymic epithelial cells promotes thymocyte positive selection and differentiation by repression of Shh

Abstract

Similar works

Full text

Available Versions

UCL Discovery