Thymosin b4 Induces Folding of the Developing Optic Tectum in the Chicken (Gallus Domesticus) Hans-Georg Wirsching, 1 * Oliver Kretz, 2 Gabriela Morosan-Puopolo, 1,3 Petya Chernogorova, 1 Carsten Theiss, 3 and Beate Brand-Saberi 1,3 * 1 Department of Molecular Embryology, University of Freiburg, D-79104 Freiburg, Germany 2 Department of Neuroanatomy, University of Freiburg, D-79104 Freiburg, Germany 3 Department of Anatomy and Molecular Embryology, Ruhr University Bochum, D-44801 Bochum, Germany ABSTRACT Thymosin b4 (Tb4) is a highly conserved G-actin binding polypeptide with multiple intra- and extracellular func- tions. While stem-cell activation as well as promotion of cell survival and migration by Tb4 have been investi- gated in various in vitro and in vivo studies, there are few data on the implications of Tb4 in brain develop- ment. In the present study we analyzed Tb4 expression in the developing optic tectum of the chicken (Gallus domesticus) and performed in ovo retroviral transduc- tion and plasmid electroporation for overexpression and knockdown of Tb4. We found marked Tb4 expression in the tectal plate and in all neuronal layers of later devel- opmental stages, but not in the ventricular zone where neural stem cells reside and divide. Knockdown of Tb4 inhibited growth of Tb4-depleted hemispheres, whereas overexpression of Tb4 led to the production of neuroe- pithelial folds resembling gyri and sulci, which are not normally present in avian brains. The mechanism yield- ing enhanced growth of Tb4 overexpressing hemi- spheres involved enhanced proliferation, thus indicating an impact of Tb4 on the neural stem cell and/or pro- genitor cell population. In summary, we found that due to its effects on proliferation, Tb4 expression has a large impact on neuroepithelial and macroscopic brain development. J. Comp. Neurol. 520:1650–1662, 2012. V C 2011 Wiley Periodicals, Inc. INDEXING TERMS: brain development; actin binding polypeptide; neural stem cell proliferation; neuroepithelium Like the mammalian neocortex (NC), the chicken optic tectum (OT) is anatomically and functionally organized in horizontal layers and vertical columns (Gray et al., 1990). Consequently, NC and OT share basic developmental mechanisms, including proliferation predominantly within the ventricular zone, long-distance tangential migration, or migration within a functional column along radial glial processes (Gray et al., 1988; Gray and Sanes, 1991, 1992). However, convolutions of the brain surface—with crests known as gyri and grooves called sulci—normally occur exclusively in higher mammals as a result of the phylogenetic expansion of the cortical surface (Finlay and Darlington, 1995; Clark et al., 2001). During evolution a 1,000-fold increase in cortical surface occurred from mouse to human, as opposed to merely an 2-fold increase of cortical thickness (Rakic, 1988). To date, little is known about the molecular basis of horizontal expan- sion of the neuroepithelium (NE), but nuclear b-catenin appears to be a crucial downstream player (Chenn and Walsh, 2002; Falk et al., 2008). Thymosin b4 (Tb4) is a highly conserved and abun- dantly expressed 43 amino acid polypeptide with multiple intra- and extracellular functions. Originally thought to be a thymic hormone, Tb4 was found to be the major actin sequestering polypeptide in various cell types (Safer et al., 1991). Since the discovery of an autocrine and paracrine proangiogenetic effect of Tb4 in vitro (Grant et al., 1999), various Tb4 functions beyond actin seques- tering have been discovered, including stem-cell activa- tion (Philp et al., 2004; Smart et al., 2007), promotion of cell survival and migration (Bock-Marquette et al., 2004), as well as enhancement of invasiveness and metastatic Current address for Hans-Georg Wirsching: Laboratory of Molecular Neuro-Oncology, Department of Neurology, University Hospital Zu¨rich, CH-8008 Zu¨rich, Switzerland. Grant sponsor: GRK; Grant number: 1104 (to B.B.S.). *CORRESPONDENCE TO: Beate Brand-Saberi, Ruhr-Universita ¨t Bochum, Institut fu¨r Anatomie, Abteilung fu ¨r Anatomie und Molekulare Embryologie, Universita ¨tsstrabe 150, Geba ¨ude MA, Ebene 5, Raum 161, D-44801 Bochum, Germany. E-mail: beate.brand-saberi@rub.de V C 2011 Wiley Periodicals, Inc. Received May 24, 2010; Revised December 30, 2010; Accepted November 20, 2011 DOI 10.1002/cne.23004 Published online November 25, 2011 in Wiley Online Library (wileyonlinelibrary.com) 1650 The Journal of Comparative Neurology | Research in Systems Neuroscience 520:1650–1662 (2012) RESEARCH ARTICLE