Thrombospondin-4 and matrix three-dimensionality in axon outgrowth and adhesion in the developing retina Erin Tolhurst Dunkle a , Frank Zaucke b , Dennis O. Clegg a, * a Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106, USA b Center for Biochemistry, Medical Faculty, University of Cologne, Cologne D-50931, Germany Received 29 July 2006; accepted in revised form 12 December 2006 Available online 4 January 2007 Abstract Thrombospondin-4 (TSP-4), a large pentameric glycoprotein of the extracellular matrix, has been described as a neurite outgrowth-promoting molecule. However, the means by which TSP-4 promotes neurite outgrowth in the developing eye is unclear. Here we show that TSP-4 is present at the appropriate time in development and displays a localization pattern within the developing mouse retina consistent with a role in retinal ganglion cell (RGC) neurite outgrowth. Furthermore, results indicate that while TSP-4 alone does not support adhesion or neurite extension, it enhances the ability of laminins to promote adhesion and neurite outgrowth of embryonic retinal cells. The mechanism of enhancement is, in part, based on the ability of TSP-4 to enhance the three-dimensionality and/or clustering of laminins within the substrate matrix. These results support a model where TSP-4 acts as an organizer of adhesive and axon outgrowth-promoting molecules in the ECM to optimize retinal ganglion cell responses. Published by Elsevier Ltd. Keywords: thrombospondin-4; extracellular matrix; retinal development; neurite outgrowth; adhesion; three-dimensionality 1. Introduction The cells of the neural retina are heavily influenced by the surrounding extracellular matrix (ECM), and this influence is crucial for proper development. In order to stratify properly and make the connections necessary to establish vision, retinal neurons must sense and respond to a vast array of positive, negative, and instructive cues present in the surrounding envi- ronment. Positive cues would include permissive or attractive molecules, while negative cues could be inhibitory or repul- sive (Stoeckli and Landmesser, 1998; Clegg et al., 2000, 2003). These cues may be secreted, membrane bound, or com- ponents of the ECM (Mueller, 1999; Bonner and O’Connor, 2001; Monnier et al., 2002; Oster et al., 2004). The thrombospondins are a highly related family of se- creted glycoproteins expressed throughout the developing ner- vous system. The thrombospondin family is composed of five mammalian members: TSP-1, TSP-2, TSP-3, and TSP-4 and TSP-5/COMP (cartilage oligomeric matrix protein), in addi- tion to the multitude of fish and invertebrate thrombospondins that have been identified (Adams et al., 2003; McKenzie et al., 2006). Thrombospondins are divided into two groups, A and B, based on their domain structure and multimerization status. Characteristic of ECM proteins, thrombospondins have a mul- tidomain structure containing several types of repeating se- quence elements (Hohenester and Engel, 2002). Common to all thrombospondins is the C-terminal cassette composed of Type 2 (EGF-like) repeats, Type 3 calcium binding repeats and a globular C-terminus domain (Adams, 2004). The sub- group A thrombospondins, TSP-1 and TSP-2, also contain a von Willebrand factor type C domain and thrombospondin Type 1 (TSR) repeats. With the exception of TSP-5 (COMP), the thrombospondins have a globular N-terminal * Corresponding author. Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA. Tel.: þ1 805 893 8490; fax: þ1 805 893 2005. E-mail address: clegg@lifesci.ucsb.edu (D.O. Clegg). 0014-4835/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.exer.2006.12.014 Experimental Eye Research 84 (2007) 707e717 www.elsevier.com/locate/yexer