Identification of lynx2, a novel member of the ly-6/neurotoxin superfamily, expressed in neuronal subpopulations during mouse development

Abstract

We isolated a new gene which shares all the features of the Ly-6/neurotoxin superfamily, from gene organization to predicted 3D structure. As it is preferentially expressed in the nervous system, we called this gene lynx2, by analogy with lynx1, a nAChR modulator. In embryonic and postnatal mouse, lynx2 is expressed in postmitotic central and peripheral neurons. These include subpopulations of motor neurons, sensory neurons, interneurons and neurons of the autonomous nervous system. In addition, lynx2 is transiently expressed around the growing nerves in the limb bud. Comparison of its spatio-temporal expression pattern with that of two other members of this family, lynx1 and ly-6h, shows that these genes are detected both in distinct and overlapping neuron populations.

Introduction

One of the fundamental issues in the field of developmental neuroscience is to understand the mechanisms that control the identity of distinct classes of neurons as well as the specificity of the connections they elaborate. In adult organisms, the mature identity of a neuron, which underlies the functional unit of a neural circuit, is represented by its characteristic features that include soma position, axonal projection pattern, elaboration of dendritic arborization, expression of specific ion channels and neurotransmitter receptors and production of the appropriate neurotransmitter. Many of these specific traits are acquired and coordinately regulated during embryonic development (for review, see Edlund and Jessell, 1999, Jessell, 2000).

Recent genetic studies of vertebrate spinal cord development have identified a number of genes that regulate the expression of these unique phenotypes. The process of specifying motor neuron identity can be thought of as encompassing three stages generating a somatotopic representation of the periphery. First, the specification of generic motor neuron identity leads to the generation of motor neurons. Second, the soma of functionally related groups of motor neurons that are destined to share common projection targets settle in longitudinally oriented columns in the spinal cord. Concomitantly, their axons project towards their target regions. Third, the cell bodies of motor neurons that innervate the same muscle form clusters known as motor pools and both pre- and postsynaptic connections are made. Each of these steps involves extracellular signals that regulate intrinsic determinants of motor neuron identity, which together define and shape motor neuron development in a sequential fashion (for review, see Jessell, 2000).

In addition to this genetic control, spinal cord development relies on activity-dependent mechanisms that configure embryonic spinal circuits. Though the genetic framework has been largely documented, relatively few studies have focused on activity-dependent mechanisms. At early developmental stages, when motor axons have just exited the spinal cord, neurons within the spinal cord generate spontaneous patterns of activity whose frequencies are different between distinct motor pools (Milner and Landmesser, 1999). These early episodes of spontaneous activity, which precede invasion by axons of the limb bud, are driven by glycinergic, γ-aminobutyric acid (GABA)ergic and cholinergic transmission (Borodinsky et al., 2004, Hanson and Landmesser, 2003, Milner and Landmesser, 1999). Blocking or slowing down this bursting activity leads to dorsal–ventral guidance errors in the peripheral hindlimb and alterations in expression of several guidance/adhesion molecules (Hanson and Landmesser, 2004). In addition, cholinergic transmission is required for proper development of locomotor circuits during a transient period of embryonic development (Myers et al., 2005). Taken together, these results support previous findings that have shown a role for acetylcholine in the modulation of motor output (Perrins and Roberts, 1995).

Similarly to exogenous toxins which bind to and regulate the function of nicotinic acetylcholine receptors (nAChRs), two proteins called SLURP-1 and Lynx1, which are expressed in epidermis and postnatal nervous system, respectively, act as endogenous prototoxins to modulate nAChRs in vitro and in vivo (Chimienti et al., 2003, Ibanez-Tallon et al., 2002, Ibanez-Tallon et al., 2004, Miwa et al., 1999). SLURP-1 and Lynx1 are members of the Ly-6/neurotoxin superfamily (SF), a name coming from an evolutionary relationship proposed between the snake venom neurotoxins and the mammalian ly-6 genes that have long been studied in the immune system (Gumley et al., 1995). Ly-6 is a multigene family encoding cell surface proteins mainly expressed on cells of hemopoietic origin. In the immune system, they are mainly involved in cellular interactions and T cell activation (Gumley et al., 1995).

We screened a cDNA library from embryonic ventral spinal cord and isolated a gene that belongs to the Ly-6/neurotoxin SF. Based on its structural similarity with Lynx1, which is also expressed in discrete neuronal populations, we assigned the name lynx2 to this novel ly-6 gene. Ontogeny of lynx2 expression in the nervous system allowed us to associate lynx2 with specific neuronal subpopulations in mouse, both at embryonic and postnatal stages. We compared the expression of lynx2 with two other members of this SF previously known to be expressed in the nervous system and show that they are expressed in both distinct and overlapping neuronal populations.