Loss of Gli3 and Shh Function Disrupts Olfactory Axon Trajectories CURTIS WILLIAM BALMER AND ANTHONY-SAMUEL LAMANTIA * Department of Cell & Molecular Physiology, Curriculum in Neurobiology and University of North Carolina Neuroscience Center, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, North Carolina 27599 ABSTRACT The transcriptional regulator Gli3 and the secreted signal Shh influence induction, patterning, and differentiation at several sites of mesenchymal/epithelial (M/E) interaction including the limbs, heart, face, and forebrain. We asked whether loss of function of these two genes has specific consequences for early differentiation of the primary olfactory pathway— which comprises both craniofacial and forebrain structures and depends on M/E induction during initial stages of development. Loss of Gli3 or Shh function does not compromise several aspects of olfactory receptor neuron (ORN) and olfactory ensheathing cell maturation; however, directed outgrowth of ORN axons and their initial targeting to the telencephalon is altered. In Gli3 mutant extra toes-Jackson (Xt J Xt J ) embryos, ORN axons defasciculate and project aberrantly near the forebrain. They rarely enter the central nervous system, and their association with mesenchymal laminin is disrupted. In Shh / embryos, ORN axons exit a single olfactory epithelium (OE) that develops centrally within an altered mesenchymal environment in a dysmorphic proboscis. These axons project as a single nerve toward the mutant forebrain; however, their trajectory varies according to the position of the proboscis relative to the forebrain. These alterations in axon outgrowth probably reflect compromised inductive interactions in the olfactory primordia because neither Gli3 nor Shh are expressed in olfactory neurons. Thus, two genes that influence induction and subsequent differentiation of craniofacial structures and the forebrain have distinct consequences for ORN axon growth during the initial genesis of the olfactory pathway. J. Comp. Neurol. 472:292–307, 2004. © 2004 Wiley-Liss, Inc. Indexing terms: patterning; forebrain development; extracellular matrix Inductive interactions between mesenchyme and epi- thelium (M/E) influence local axis formation, cellular dif- ferentiation, and morphogenesis at several embryonic sites including the limbs, heart, branchial arches, and forebrain. In vitro analysis of early olfactory pathway development suggests that M/E induction also influences the establishment of axes within the frontonasal processes as well as subsequent olfactory receptor neuron (ORN) differentiation and axon outgrowth (Whitesides et al., 1998; LaMantia et al., 2000; Bhasin et al., 2003). The differentiation of olfactory neurons and directed out- growth of their axons appear to depend in particular on proper establishment of local medial-lateral axial coordi- nates (LaMantia et al., 2000). Thus, genetic mutations that perturb inductive interactions in the craniofacial pri- mordia and patterning of the craniofacial midline proba- bly disrupt ORN differentiation as well as assembly and targeting of the olfactory nerve in vivo. To test this hy- pothesis, we analyzed early olfactory pathway develop- ment in Shh and Gli3 homozygous null mutant mice in which M/E induction is probably disrupted and the cranio- facial midline is collapsed and expanded, respectively. In Gli3 mutant embryos (extra toes-Jackson;Xt J /Xt J ; Hui & Joyner, 1993; Schimmang et al., 1993), craniofacial morphology is severely disrupted and the forebrain lacks Grant Sponsor: National Institute of Child Health and Human Develop- ment, National Institutes of Health; Grant number: HD029178 (A.-S.L); Grant sponsor: Stanley Foundation Scholars Award (C.W.B. ). *Correspondence to: Anthony-Samuel LaMantia, Department of Cell & Molecular Physiology, Curriculum in Neurobiology and UNC Neuroscience Center, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599 Received 6 August 2003; Revised 11 December 2003; Accepted 12 De- cember 2003 DOI 10.1002/cne.20053 Published online the week of March 15, 2004 in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 472:292–307 (2004) © 2004 WILEY-LISS, INC.