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Leucine-rich repeat proteins expressed in the developing vertebrate nervous system comprise a complex, multifamily group, and little is known of their developmental function in vivo. We have identified a novel member of this group in Xenopus laevis, XlNLRR-6, and through sequence and phylogenetic analysis, have placed it within a defined family of vertebrate neuronal leucine-rich repeat proteins (NLRR). XlNLRR-6 is expressed in the developing nervous system and tissues of the eye beginning at the neural plate stage, and expression continues throughout embryonic and larval development. Using antisense morpholino oligonucleotide (MO) -mediated knockdown of XlNLRR-6, we demonstrate that this protein is critical for development of the lens, retina, and cornea. Reciprocal transplantation of presumptive lens ectoderm between MO-treated and untreated embryos demonstrate that XlNLRR-6 plays autonomous roles in the development of both the lens and retina. These findings represent the first in vivo functional analysis of an NLRR family protein and establish a role for this protein during late differentiation of tissues in the developing eye. Developmental Dynamics 235:1027–1041, 2006. © 2006 Wiley-Liss, Inc.
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Leucine-rich repeats (LRR) are found in a complex and diverse superfamily of proteins, including those involved in early embryonic patterning (Hashimoto et al.,1988), brain development, and axon pathfinding (Rothberg et al.,1988; Nose et al.,1992; Taniguchi et al.,2000; Fukamachi et al.,2001), nervous system regeneration (Ishii et al.,1996; Bormann et al.,1999), learning and memory (Bando et al.,2005), and various mammalian cancers (Almeida et al.,1998; Hamano et al.,2004). Whereas LRR-containing proteins vary greatly in their number of LRR, tertiary structure, subcellular localization, enzymatic and signaling activities, they share a repeated sequence of 20–29 amino acids rich in stereotypically placed leucines and other aliphatic residues (Kobe and Deisenhofer,1994, 1995; Hayata et al.,1998).
With the resolution of the crystal structure of the LRR-containing porcine ribonuclease inhibitor, Kobe and Deisenhofer (1994, 1995) established that each LRR forms a hydrophobic β-sheet with conserved aliphatic residues followed by a more variable, parallel α-helix. Serial placement of these β- units forms a concave, horseshoe-shaped structure with a large surface area optimal for protein–protein interactions at the adjacent β-sheets. Subsequently to this characterization, LRR-containing proteins have been implicated in protein–protein interactions such as adhesion and intercellular signaling (Taniguchi et al.,1996; Fukamachi et al.,2001, 2002).
A family of LRR-containing proteins was first identified in the mouse as having a characteristic 11–12 LRR, flanked at the N-terminal and C-terminal sides with stereotypically placed cysteine residues (amino flanking region, NFR; carboxy flanking region, CFR), and also containing an immunoglobulin type C2 (IgC2) loop and a single transmembrane domain with a very short intracellular carboxy-terminal region. Some of these proteins also contain a fibronectin type 3 (FnIII) domain, an RGD integrin binding motif, and a clathrin-mediated endocytosis motif (see Fig. 1A). These proteins were named neuronal leucine-rich repeat (NLRR) proteins, based on their recovery from a neonatal mouse brain cDNA library (Taguchi et al.,1996). Members of the NLRR protein family have since been identified in the frog Xenopus, zebrafish, rat, and human, with predicted protein homologs in many other vertebrates. See Table 1 for selected members of this family.
Figure 1. Characteristic neuronal leucine-rich repeat proteins (NLRR) protein features and inferred XlNLRR-6 protein sequence. A: The schematic diagram illustrates the conserved features among the known NLRR proteins. Not all members of the NLRR family have every illustrated domain; see Table 1. N-terminal (extracellular) domains are toward the left; C-terminal domains are toward the right. S, signal peptide; NFR, amino-flanking region with stereotypically placed cysteine residues; LRR, leucine-rich repeats; CFR, carboxy-flanking region with stereotypically placed cysteine residues; IgC2, immunoglobulin type C2 domain; FnIII, fibronectin type III domain; T, transmembrane helix. Also indicated are the positions of the RGD sequence in the eighth LRR of NLRR-3 proteins (black arrowhead) and the clathrin-mediated endocytosis motif of NLRR-1, -3, and -6 proteins (white arrowhead). B: The predicted protein sequence of XlNLRR-6 is shown. The LRR regions are aligned, illustrating the consensus sequence of xLxxLxxLxLxxNxL. In some cases, other aliphatic residues substitute for leucines (e.g., valine or isoleucine). Residues that differ from the consensus residues are indicated in lighter shading. Also indicated are the IgC2 domain (gray shading), transmembrane helix (double underline), and clathrin-mediated endocytosis motif (single underline).
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Table 1. Features of NLRR Protein Family Members
|NLRR-1||M. musculus||716 aa||12||+||−||−||+||D45913||Taguchi et al.,1996|
| ||X. laevis||718 aa||12||+||+||−||+||AB014462||Hayata et al.,1998|
| ||H. sapiens||716 aa||11||+||−||−||+||BC034947||Hamano et al.,2004|
|NLRR-2 (GAC1)b||M. musculus||730 aa||11||+||−||−||−||D49375||Taguchi et al.,1996|
| ||H. sapiens||713 aa||11||+||−||−||−||AY358290||Hamano et al.,2004|
|NLRR-3||M. musculus||707 aa||11||+||+||+||+||D49802||Taniguchi et al.,1996|
| ||R. norvegicus||707 aa||11||+||+||+||+||AF291437||Fukamachi et al.,2001|
| ||H. sapiens||708 aa||11||+||+||+||+||AB060967||Hamano et al.,2004|
|NLRR-4||M. musculus||735 aa||8||−||+||−||−||BC056458||Bando et al.,2005|
|NLRR-5||H. sapiens||713 aa||11||+||−||−||−||CAI14096||Hamano et al.,2004|
|proposed: NLRR-6||D. rerio||744 aa||12||+||−||−||+||n/a||Bormann et al.,1999|
| ||X. laevis||739 aa||12||+||−||−||+||DQ315790||Wolfe et al.,2004; this study|
Additional vertebrate proteins have been described, which share some of the NLRR protein features shown in Figure 1A but appear to be less closely related. These proteins have been variously named leucine-rich repeat neuronal (LRRN or LERN) and leucine-rich repeat transmembrane neuronal (LRRTN or LRRTM) proteins; members of these groups are also expressed in the nervous system (Carim-Todd et al.,2003; Lauren et al.,2003).
Whereas little in vivo functional examination has been conducted on NLRR proteins, their roles in nervous system development have been predicted largely based on expression data and in vitro studies. Mouse NLRR-1, -2, and -3 (see Table 1) are all expressed during nervous system development, and expression continues in discrete regions of the adult brain (Taguchi et al.,1996; Taniguchi et al.,1996). In Xenopus, NLRR-1 is expressed in the central nervous system (CNS) ventricular zone of the early tadpole, suggesting a role in neural layer formation within the brain and spinal cord (Hayata et al.,1998).
Rat NLRR-3 has been examined in vitro and is implicated in long-term potentiation of epidermal growth factor (EGF) signaling. NLRR-3 transcription in fibrosarcoma cells was up-regulated after EGF stimulation and also in the presence of the consitutively active small G-protein ras, while transcription decreased in the presence of mitogen-activated protein kinase (MAPK) inhibitors, suggesting that NLRR-3 transcription is downstream of EGF by means of a MAPK-dependent pathway (Fukamachi et al.,2001). Further examination revealed that NLRR-3 facilitates internalization of the EGF ligand/receptor complex, which continues to transduce intracellular signal after internalization (Fukamachi et al.,2002). The authors concluded that EGF ligand/receptor complexes cluster with NLRR-3 proteins, and the NLRR-3 cluster encourages the formation of a clathrin-coated vesicle by means of a nine-residue motif in its intracellular domain (YPPLI[N or S]LWE). Because three groups of NLRR proteins contain this same motif (see Table 1), the roles of these proteins, in part, also may involve internalization of signaling molecules.
In addition to roles in cell signaling and neural development, three NLRR proteins have been identified as playing a role in vertebrate neural regeneration. Mouse NLRR-3 transcription is up-regulated after cortical injury, specifically in layer III of the cortex, whereas NLRR-1 and -2 do not demonstrate this up-regulation. Thus, NLRR-3 may be associated with neuronal regeneration and reconstruction in the cerebral cortex (Ishii et al.,1996). In zebrafish, zfNLRR demonstrates prominent expression in the developing eye and CNS and expression is dramatically up-regulated in neurons whose axons regenerate after damage to the optic nerve and spinal cord (Bormann et al.,1999). A newly identified Xenopus expressed sequence tag (EST) tentatively named W006, with significant sequence similarity to other NLRR proteins, shows similar expression in the developing eye and brain, with up-regulation in the regenerating lens and hind limb (Wolfe et al.,2004). zfNLRR and W006 have a high sequence similarity to each other, indicating a possible shared role in development and regeneration (see below).
We have cloned the full length of the W006 gene and examined its relationship to other NLRR family members, as well as to other neuronally expressed LRR-containing proteins. Based on these analyses, W006 belongs in a new NLRR group with zfNLRR, which we have named NLRR-6. The timing and organ specificity of W006 expression has been characterized as beginning at neurulation, and larval expression is restricted to the developing lens, retina, and brain. As there is no previous in vivo work examining the role of NLRR proteins during development, we set out to establish the role of W006 in eye development, where the transcript is readily detectable in the lens and retina. Using antisense morpholino oligonucleotide-mediated knockdown of W006, we have established that W006 is necessary for proper differentiation of the lens and retina and that these roles are autonomous to each tissue.