Laminin-5 promotes neurite outgrowth from central and peripheral chick embryonic neurons Brian Culley a , James Murphy a , Joe Babaie a , Diane Nguyen a , Amy Pagel a , Patricia Rousselle b , Dennis O. Clegg a, * a Neuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA b Institut de Biologie et Chimie des Proteines, Lyon, France Received 23 October 2000; received in revised form 12 January 2001; accepted 19 January 2001 Abstract Laminin-5 (Ln-5) is an essential component of epithelial basal laminae that is also expressed in the developing nervous system. Here we use a convenient, simple and reproducible in vitro ¯uorescent assay to assess the neurite outgrowth promoting activity of puri®ed Ln-5. Embryonic chick neurons from dorsal root ganglia, ciliary ganglia, and (to a lesser extent) retina extended neurites on Ln-5, but the neurite outgrowth promoting activity was not as great as that of Ln-1 or Ln-2. Neurons from diencephalon, telencephalon, and spinal cord did not respond to Ln-5. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Laminin-5; Neurite outgrowth assay; Calcein AM; Extracellular matrix The correct wiring of the nervous system is thought to be choreographed in part by molecules in the microenviron- ment that stimulate the extension of axons, such as the laminins [10]. Laminins are a family of heterotrimeric ECM glycoproteins that induce adhesion, axon and dendrite outgrowth, and also arrest of growth cone movement. [2,4,7,14,15]. Laminin 5, consisting of the a3A, b3, and g2 chains, is somewhat unusual in that it forms a rod-like structure, rather than a cross [1,7]. Ln-5 subunits are expressed in regions that receive sensory innervation, such as skin, and transient expression has been noted in the developing central nervous system, including the retina [13]. Earlier studies have demonstrated that puri®ed Ln-5 promotes neurite outgrowth from neural cell lines via a complex of integrin a3b1 and the tetraspanins CD151 and CD81 [16,17]. However, whether Ln-5 supports neurite outgrowth from primary neurons has not been investigated. Here we use a ¯uorescent neurite outgrowth assay in 96 well plates to demonstrate that Ln-5 stimulates neurite outgrowth from some but not all embryonic chick neurons. Murine Ln-1 and Human Ln-2 (Merosin) were purchased from Sigma (St. Louis, MO, USA) and Gibco-BRL (Gaithersburg, MD, USA), respectively. Human Ln-5 was puri®ed as described in [16]. The function blocking anti-b1 integrin antibody CSAT was puri®ed as described in [3]. Non immune mouse IgG was purchased from Sigma. Calcein AM was from Molecular Probes (Eugene, Oregon, USA) and eggs were from Rosemary Farms (Santa Maria, CA, USA). Primary chick embryonic neurons (see Table 1) were cultured in 100 ul volumes as described in [3]. For cultures of dorsal root ganglion neurons, 7S nerve growth factor (NGF; Sigma) was added at a concentration of 2 mg/ml. Ninety-six well plates were coated as described in [3], except they were blocked with 5% non-fat dry milk. After neurons were cultured overnight, 5 ml of 20 uM calcein AM [19] was added to each well. In some experiments, 1 ml of 40 mM ethidium bromide (Molecular Probes) was also added to the well to visualize dead cells. Plates were returned to the incubator for 1 h, and then immediately photographed using an inverted Olympus IMT-2 microscope. Neurite outgrowth was quanti®ed as described in [5]. For each determination, at least 50 cells were analyzed using four to six micrographs from two to three separate wells. To determine if Ln-5 supports neurite outgrowth to the same extent as Ln-1, we cultured embryonic day 7 chick DRG neurons on Ln-1 and Ln-5, and stained them with calcein AM (Fig. 1). The calcein AM dye completely ®lls Neuroscience Letters 301 (2001) 83±86 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)01615-9 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 11-805-893-8490; fax: 11-805- 893-2005. E-mail address: clegg@lifesci.ucsb.edu (D.O. Clegg).