Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos LORI HARTNETT 1 , CATHERINE GLYNN 1 , CATHERINE M. NOLAN 2 , MAURA GREALY 3 and LUCY BYRNES* ,1 1 Department of Biochemistry, National University of Ireland, Galway, Ireland, 2 School of Biology and Environmental Science, University College Dublin, Ireland and 3 Department of Pharmacology and Therapeutics, National University of Ireland, Galway, Ireland ABSTRACT The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf- 2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti- apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf- 2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome. KEY WORDS: zebrafish, IGF-2, neural, cardiovascular, development Introduction Insulin-like growth factor-2 (IGF-2) is a single chain polypep- tide that acts as a foetal promoter of cell growth, survival and differentiation. In mammals, IGF-2 can bind to two receptors: the IGF-1 receptor (IGF-1R) and the IGF-2 receptor (IGF-2R). The IGF-1R belongs to the tyrosine kinase receptor superfamily. It is a heterotetrameric transmembrane protein, which mediates most of the effects of IGF-1 and IGF-2. The IGF-2R is a single-chain transmembrane protein and there is no evidence of a role for the IGF-2R in transducing IGF-2 signals. The IGF family is made more complex by the presence of IGF binding proteins (IGFBPs). Int. J. Dev. Biol. 54: 573-583 (2010) doi: 10.1387/ijdb.092922lh THE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY www.intjdevbiol.com *Address correspondence to: Dr. Lucy Byrnes. Department of Biochemistry, National University of Ireland, Galway, Ireland. Fax: +353-91-525700. e-mail: lucy.byrnes@nuigalway.ie - Web: http://www.nuigalway.ie/biochemistry/staff/byrnes/index.html Supplementary Material for this paper (tables, figures and 3 movies) is available at: http://dx.doi.org/10.1387/ijdb.092922lh Accepted: 20 April 2009. Final author-corrected PDF published online: 4 September 2009. Edited by: Edward de Robertis ISSN: Online 1696-3547, Print 0214-6282 © 2009 UBC Press Printed in Spain Abbreviations used in this paper: BMP, bone morphogenetic protein; DN-IGF- 1R, dominant negative insulin-like growth factor-1 receptor; eln2, tropoelastin 2; GSK3, glycogen synthase kinase 3; hpf, hours post fertilisation; IGF, insulin-like growth factor; IGFBP, IGF binding protein; IGF-1R, IGF-1 receptor; MAPK, mitogen activated protein kinase; PI3K, phosphatidylinositol- 3 kinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. These act to modulate the actions of IGFs by either inhibiting or augmenting their availability. The majority of IGF signals are transduced by ligand binding to the IGF-1R. This triggers autophosphorylation of the receptor, which ultimately leads to the