A novel role for Glucocorticoid-Induced TNF Receptor Ligand (Gitrl) in early embryonic zebrafish development LYNN D. POULTON 1,a,c , KATHLEEN F. NOLAN* ,1,a , CORINA ANASTASAKI 2 , HERMAN WALDMANN 1,b and E. ELIZABETH PATTON 2,b 1 Sir William Dunn School of Pathology, University of Oxford, Oxford, and 2 Institute of Genetics and Molecular Medicine, MRC Human Genetics Unit and The University of Edinburgh, Edinburgh, UK ABSTRACT Tumour necrosis factor ligand and receptor superfamily (TNFSF and TNFRSF) members have diverse and well-studied functions in the immune system. Additional, non- immunological roles, such as in the morphogenesis of bone, tooth, hair and skin have also been described for some members. GITRL and its receptor GITR are well-described as co-regulators of the mammalian immune response. Here, we describe the identification and cloning of their zebrafish homologues and demonstrate a novel role for the ligand, but not the receptor, in early vertebrate development. The assignment of zebrafish Gitrl and Gitr was supported by homology and phylogenetic analysis. The ligand exhibited an oscillating pattern of mRNA expression during the first 36 hours post fertilization, during which time gitr mRNA was not detected, and morpholino oligonucleotide-mediated knock-down of gitrl, but not of gitr, resulted in disruption of early embryogenesis, most clearly revealed during gastrulation, which corresponded to the earliest peak in gitrl mRNA expression (5.25-10 hpf). We found Stat3 signalling to be altered in the gitrl-morphants, suggesting that one possible role for Gitrl during embryogenesis may be modulation of Jak/Stat signalling. KEY WORDS: GITRL, GITR, development, zebrafish, Stat3 Introduction The TNF and TNFR superfamilies are receptor-ligand signal- ling molecules that regulate a variety of pathways, but have primarily been associated with the maintenance and regulation of the immune system. One of these, the glucocorticoid-induced TNF receptor (GITR), in addition to being expressed on activated effector T cells, was also highlighted in a screen for genes expressed by regulatory T cells (Zelenika et al., 2002). Regulatory T cells engage in the suppression of activated effector T cells, including self-reactive T cells, thereby controlling the immune response and helping to prevent autoimmunity. The ligand for GITR, GITRL, is constitutively expressed on antigen presenting cells. Its expression is transiently increased in response to inflam- matory stimuli, and engagement of GITR by GITRL leads to both dampening of the suppressive effects of regulatory T cells, and to co-activation of effector T cells (Tone et al., 2003). The net Int. J. Dev. Biol. 54: 815-825 (2010) doi: 10.1387/ijdb.082841lp THE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY www.intjdevbiol.com *Address correspondence to: Dr. Kathleen F Nolan. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK. Fax: +44 (0)1865-275501. e-mail: kathleen.nolan@path.ox.ac.uk Notes: a L.D.P and K.F.N contributed equally to this work. b H.W. and E.E.P. contributed equally to this work. c Present Address: Arana Therapeutics, Epping Rd, Macquarie Park NSW 2113 Australia Accepted: 2 June 2009. Final author-corrected PDF published online: 3 July 2009. ISSN: Online 1696-3547, Print 0214-6282 © 2009 UBC Press Printed in Spain Abbreviations used in this paper: dpf, days post-fertilization; EDA, ectodysplasin; hpf, hours post fertilization; GITR, glucocorticoid-induced TNF receptor; GITRL, glucocorticoid-induced TNF receptor ligand; MO, morpholino oligonucleotide; TNFSF, tumour necrosis factor superfamily; TNFRSF tumour necrosis factor receptor superfamily. effective outcome is strong activation of the immune response, and indeed, antibody-mediated stimulation of GITR can induce autoimmunity (Shimizu et al., 2002). The potential to modulate immune regulation through GITRL-GITR signalling has made this pathway an interesting candidate for study in cancer immuno- therapy and in models of persistent infection. In mice, targeted disruption of the GITR gene results in T cell hypersensitivity to CD3 activation, leading to hyperproliferation and heightened sensitivity to activation-induced cell death (Ronchetti et al., 2002), as well as altered responses to Candida