Analysis of Lrrn1 expression and its relationship to neuromeric boundaries during chick neural development
© Andreae et al.; licensee BioMed Central Ltd. 2007
Received: 26 March 2007
Accepted: 31 October 2007
Published: 31 October 2007
The Drosophila leucine-rich repeat proteins Tartan (TRN) and Capricious (CAPS) mediate cell affinity differences during compartition of the wing imaginal disc. This study aims to identify and characterize the expression of a chick orthologue of TRN/CAPS and examine its potential function in relation to compartment boundaries in the vertebrate central nervous system.
We identified a complementary DNA clone encoding Leucine-rich repeat neuronal 1 (Lrrn1), a single-pass transmembrane protein with 12 extracellular leucine-rich repeats most closely related to TRN/CAPS. Lrrn1 is dynamically expressed during chick development, being initially localized to the neural plate and tube, where it is restricted to the ventricular layer. It becomes downregulated in boundaries following their formation. In the mid-diencephalon, Lrrn1 expression prefigures the position of the anterior boundary of the zona limitans intrathalamica (ZLI). It becomes progressively downregulated from the presumptive ZLI just before the onset of expression of the signalling molecule Sonic hedgehog (Shh) within the ZLI. In the hindbrain, downregulation at rhombomere boundaries correlates with the emergence of specialized boundary cell populations, in which it is subsequently reactivated. Immunocolocalization studies confirm that Lrrn1 protein is endocytosed from the plasma membrane and is a component of the endosomal system, being concentrated within the early endosomal compartment.
Chick Lrrn1 is expressed in ventricular layer neuroepithelial cells and is downregulated at boundary regions, where neurogenesis is known to be delayed, or inhibited. The timing of Lrrn1 downregulation correlates closely with the activation of signaling molecule expression at these boundaries. This expression is consistent with the emergence of secondary organizer properties at boundaries and its endosomal localisation suggests that Lrrn1 may regulate the subcellular localisation of specific components of signalling or cell-cell recognition pathways in neuroepithelial cells.
Early neural development in vertebrates proceeds via the progressive regionalization of the neuroepithelium . In some cases, most prominently the hindbrain and diencephalon, nascent regions become compartmented by differences in cell-cell affinity, which prevents the mixing of cells between adjacent regions. Cells lying at the boundaries between compartments may later form specialized signaling centres, or local (or secondary) organizers, that inform neigbouring cells about their position and fate. An important function of inter-compartment lineage restriction is that it stabilizes the position of the signal source and maintains a straight interface with the sink, both of which are crucial to the formation of consistent morphogen gradients.
Specialized local organizers pattern both the dorsoventral (DV) and the anteroposterior (AP) axes of the neural tube. The floor plate, at the ventral midline of the neural tube, has organizer activity through the ventralizing actions of Sonic hedgehog (Shh) and the transforming growth factor (TGF)β family member Nodal . Similarly, at the dorsal midline, the roof plate exerts a dorsalizing activity by producing TGFβs and Wnts . Along the AP axis, several boundaries have been characterized as local organizers. These include: the anterior neural border (ANB; also known as the anterior neural ridge in amniotes or Row-1 in zebrafish) at the anterior margin of the neural plate , which signals through secreted Wnt antagonists and the fibroblast growth factor (FGF)8 [5–7]; the zona limitans intrathalamica (ZLI), which signals through Shh and, perhaps, Wnt8b [8–10]; the midbrain-hindbrain boundary (MHB), which signals through the actions of FGF8 and Wnt1 ; and inter-rhombomere boundaries, which signal through Wnt1 [12, 13].
In the chick embryo hindbrain, inter-rhombomere boundaries begin to form at Hamburger and Hamilton (HH) stage 9  and develop into ridges or thickenings on the ventricular surface of the neural tube where the rate of mitosis is reduced. These ventricular ridges are associated with disrupted interkinetic nuclear migration , with cells being deflected into fan-shaped arrays on the apical-basal axis. Rhombomere boundary cells share a number of characteristics with radial glia, such as increased expression of the transcription factor Pax6 and the intermediate filament protein vimentin . A similar phenomenon is observed at major boundaries in the forebrain, such as the diencephalic-mesencephalic boundary (DMB) and the prethalamic-thalamic boundary (ZLI) . The conspicuous accumulation of axonal growth promoting extracellular matrix components highlights the subsequent function of some, but not all, boundaries as conduits for axon tracts [1, 17–19].
Cells from neighbouring rhombomeres do not intermingle if the boundary between them is ablated microsurgically , or if boundary cell formation is blocked by the application of retinoic acid , indicating the lineage restriction between adjacent compartments is not imposed by a simple physical barrier to cell mixing, at least in the hindbrain. However, an early feature of rhombomere boundaries is a relative increase in the volume of extracellular space compared to rhombomere bodies, which is likely to be a consequence of the immiscibility between cells of neighboring rhombomeres due to cell-cell affinity differences [22–24]. Indeed, it has been known for some time that cells from alternate rhombomeres have differential affinities for one another , suggesting that this property could underlie the process of boundary formation. More recently, the molecular mechanisms responsible for this differential affinity have been identified following the observation that membrane associated Eph receptor tyrosine kinases and their ligands, the ephrins, have complementary expression in the odd and even numbered rhombomeres: boundary formation appears to be initiated by repulsive Eph-ephrin interactions at the interfaces between rhombomeres [24–26], while additional Eph-dependent adhesive mechanisms may act within rhombomeres to reinforce individual rhombomere integrity .
The mechanisms by which boundary cells themselves are specified are less clear, but are likely to involve signaling between rhombomeres. Boundary cells begin to express high levels of the glycosyl transferase radical fringe (Rfng), a likely regulator of Notch activation, whilst adjacent non-boundary cells express high levels of the Notch ligands DeltaA and DeltaD. Ectopic activation of the Notch pathway cause cells to segregate to the boundary. Moreover, inhibition of Notch signaling has an opposite effect, where cells sort away from the boundary. Thus, a high level of sustained Notch activation at the boundary appears to cause cells to adopt a boundary cell fate . Rfng is also required for boundary cell expression of Wnt1, which regulates Delta and proneural gene expression in non-boundary zones, suppressing boundary cell markers [12, 13]. Thus, boundary cells play a crucial role in coordinating the process of neurogenesis in the hindbrain, but their role in other brain regions is less well understood.
Boundary formation between dorsal (D) and ventral (V) compartments in the Drosophila wing imaginal disc shares a number of striking similarities with that in the vertebrate hindbrain. Early DV cell affinity differences, in concert with Notch signaling, establish the compartment boundary, which acts as a signaling centre through the action of wingless (Wg) to coordinate growth and patterning of the wing blade. D cell identity is specified by the LIM-homeodomain transcription factor apterous (Ap), which confers an affinity difference between D and V cells to restrict cell mixing at the DV boundary . D cell affinity characteristics are conferred by Tartan (TRN) and Capricious (CAPS), two closely related type-I transmembrane leucine-rich repeat (LRR) proteins activated by Ap . The establishment and maintenance of the affinity boundary appears to be only a transient function of Trn/Caps, which have been postulated to act as ligands for an unidentified cell surface receptor expressed on D cells .
We postulated that vertebrate LRR proteins might be involved in establishing compartment boundaries and identified the Leucine-rich repeat neuronal (Lrrn) family as being most closely related to TRN/CAPS based on sequence conservation. This three-member gene family consists of single-pass transmembrane proteins with conserved domain architecture.
Mouse Lrrn1 and Lrrn2 (first designated as neuronal leucine-rich repeat proteins (NLRR)-1 and -2, respectively) were the first members identified in a screen for LRR-containing proteins expressed in the mouse brain . Lrrn3 was isolated shortly afterwards by the same group  and subsequently in rat . Human Lrrn2 was identified independently as a gene that maps to chromosome 1 (GAC-1) and is overexpressed in gliomas, in which it is amplified . More recently, the Lrrn nomenclature has become somewhat confusing. A mouse gene identified as NLRR-4  encodes a distantly related protein (also known as C20orf75) that is not a member of the same family. Furthermore, a human gene identified as hLRRN-5  belongs to a related but different family known as LINGO/LERN/LRRN6 [38, 39]. However, the Human Gene Nomenclature Committee has recently reinstated the name LRRN2 for LRRN5.
Cloning, genomic structure and predicted protein sequence of chick Lrrn1
The deduced ORF of Lrrn1 encodes a protein of 716 amino acids that shows a remarkable degree of sequence identity to human and mouse (93%; Figure 1). Protein motif analysis reveals a domain structure characteristic of the Lrrn family. An amino-terminal signal sequence is followed by an array of 12 LRRs flanked by amino- and carboxy-terminal cysteine-rich flanking domains (LRRNT and LRRCT, respectively), a single immunoglobulin-like (Ig-like) domain and a fibronectin type III domain proximal to the single predicted transmembrane helix. The short intracellular domain (63 amino acids) contains only 2 amino acid substitutions between chick and human, both of which represent conservative changes. Within this region are two consensus endocytic sorting motifs (YXXΦ and di-leucine based) . Lrrn1 also has a consensus internal class I post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zo-1 protein (PDZ) ligand binding motif near the carboxyl terminus conforming to the consensus S/TxV/I/L [42, 43]. The high degree of sequence conservation exhibited by Lrrn1 implies that it has maintained a strict degree of functional conservation during avian-mammalian divergence. Furthermore, it suggests that interactions with other proteins, binding partners and ligands have also remained conserved.
Spatial and temporal expression of Lrrn1mRNA
Chick Lrrn1 has previously been identified by two genetic screening approaches. First, as an unknown 3' expressed sequence tag (EST) clone 2B10 isolated from a subtracted hindbrain complementary DNA (cDNA) library and expressed in the forebrain, midbrain and r2/3 of the hindbrain at HH10+ . Second, as a gene expressed in presomitc mesoderm, somites and neural tube at HH14–20 (clone 17-6G2) in a screen for signalling molecules expressed during somitogenesis . During the preparation of this manuscript, a further characterisation of clone 2B10 in the central nervous system (CNS) was published, identifying it as Lrrn1 and detailing its temporal expression in the diencephalon .
From HH12 onwards, a further marked downregulation of expression is seen around the MHB (Figure 2d). By HH14, this has expanded to include the caudal midbrain and rostral hindbrain (r1) and downregulation is also seen in the mid-diencephalic territory (likely to correspond to the future thalamus, caudal to the ZLI [8, 9]), and in the optic stalk (Figure 2e). This pattern is maintained through subsequent stages of development, but becomes more pronounced by HH18 (Figure 2f). Strong expression is maintained in the dorsal midbrain/pretectum and prethalamus, rostral to the ZLI, and telencephalon, but is downregulated at the telencephalic-diencephalic border (TDB). Whilst the striking absence of Lrrn1 expression from CNS boundary regions indicates a possible role in boundary formation or maintenance, the fact that it is also downregulated in other areas of the neuroepithelium, such as the isthmocerebellar region and the thalamus, may suggest alternative roles in proliferation or differentiation.
Given the high degree of sequence conservation between chick and mouse Lrrn1, we also compared the expression of the mouse gene at comparable developmental stages (e9.5–10.5; Figure 2g,h). Somitic expression is conserved, as both mouse and chick Lrrn1 are upregulated at the lip of the dermomyotome as cells delaminate and migrate to form the myotome (data not shown) [47, 48]). In the CNS, mouse Lrrn1 is expressed strongly in the dorsal telencephalon, albeit in a more restricted domain than in chick, and is virtually absent from the midbrain/pretectum (Figure 2f–h). Furthermore, the distribution of mouse Lrrn1 transcripts in the hindbrain is quite different (Figures 2g,h and 3e). Thus, the expression of mouse Lrrn1 does not show the same inverse relationship to boundary regions as does its chick counterpart and supports the notion that either its function is compensated for via alternative mechanisms, or it is not involved in a conserved mechanism underlying boundary formation.
Expression and localisation of Lrrn1 protein in vivo
The GST-IC antibody specifically detected a mouse Lrrn1-FLAG fusion protein in transfected cells (Figure 5b–d). We then confirmed the specificity of GST-IC in vivo by immunohistochemistry. We conducted our analyses on fresh-frozen cryosections subject to a short fixation protocol after sectioning (see Materials and methods). The distribution of staining obtained with GST-IC showed a strict congruence with the distribution of Lrrn1 mRNA by in situ hybridisation (Figure 5e,f,k,l,n,o). We did not observe any axonal labelling and staining in the hindbrain at HH17 as seen by lack of overlap with the neuronal neurofilament marker RMO-270 (Figure 5g–i). Staining was predominantly restricted to neural progenitor cells (NPCs) of the ventricular layer and was absent from rhombomere boundaries (Figure 5i–j). This indicates that, as at the RNA level, Lrrn1 is downregulated once cells leave the ventricular layer following differentiation and is absent from boundary regions. Interestingly, we noted that immunostaining within NPCs was strongest at the ventricular (apical) surface and in intensely labelled intracellular 'speckles' that have the appearance of endosomes in the hindbrain (Figure 5f). This is the first demonstration that Lrrn1 is a component of the endocytic compartment in NPCs and suggests that it may be involved in the endocytic regulation of cell adhesion or signalling mechanisms in these cells. The same distribution of staining was also seen at other sites of expression, for example, spinal cord (Figure 5k,m), retina (Figure 5n,q) and in the myotome (data not shown). We examined the temporal expression of Lrrn1 in the eye owing to the striking asymmetry in its distribution. Following the formation of the optic cup, expression becomes downregulated but remains detectable around the base of the cup where it joins the optic stalk at HH14 (Figure 5p). This domain has extended dorsalwards by HH16 but remains relatively weak and symmetrical along the AP axis. By HH18, however, expression has become upregulated strongly and exhibits an obvious ventral (strong) to dorsal (weak) gradient. Strongest expression is seen in the ventroposterior region of the retina and is absent from the lens (Figure 5n,o), which persists until at least HH21 (Figure 5q).
Endosomal localisation of Lrrn1 protein
The Lrrn family
In mammals, the Lrrn family comprises three-members, Lrrn1, Lrrn2/5 and Lrrn3 . Examination of the available genome data indicates that the same is true in avian species (JG, unpublished observation). The zebrafish genome contains two Lrrn1 genes. The first to be identified (zfNLRR) encodes a protein that is somewhat divergent from other Lrrn1s, being approximately 30 amino acids longer due to the presence of a number of insertions in the LRRCT-Ig-FnIII region . A similar atypical Lrrn1 gene has recently been described in Xenopus, named XlNLRR-6 . We have identified an additional Lrrn1-like gene in the zebrafish genome that is most similar to the other vertebrate Lrrn1 genes (Additional File 1 and Additional File 2). This points to the duplication of Lrrn1 early on in vertebrate evolution and the subsequent divergence of zfNlrr/XlNLRR-6 (which we refer to as Lrrn1b) from the archetypal Lrrn1 (Lrrn1a). The presence of Lrrn1b in both zebrafish and Xenopus, but not in mammals or birds, suggest that it has been lost in the amniote lineage, but to confirm this we must wait for more complete genome sequence data from representative reptilian and amphibian species.
Our identification and characterisation of a full-length cDNA clone for chick Lrrn1 shows that it encodes a 716 amino acid protein with a very high level of sequence identity to all vertebrate orthologues. This suggests that biochemical interactions between Lrrn1 and potential ligands/cofactors/binding partners are also likely to be highly conserved. Although comparison of the expression patterns of mouse and chicken Lrrn1 at comparable embryonic stages reveals some sites of coherence, such as the dorsal telencephalon and myotomal component of the somites, significant differences in other aspects of expression are evident between the two species. Mouse Lrrn1 does not bear the same characteristic relationship to neuromeric boundaries as chick Lrrn1 and is only weakly expressed in the midbrain and diencephalon. Neither Lrrn2 nor Lrrn3 are expressed in a way that could compensate for Lrrn1 in a redundant manner at neuromeric boundaries in mouse , suggesting that the functional role of Lrrn1 in relationship to boundaries is unique to birds, or that its function is compensated for in mammals by other factors. Although the function of Lrrn1 is currently unknown, several other families of single-pass transmembrane LRR-containing proteins have been identified (fibronectin leucine-rich transmembrane protein (FLRT)), leucine-rich transmembrane (LRRTM/LRTM) and LINGO/LRRN6/LERN (leucine-rich repeat neuronal) that could potentially fulfil this role [54–56]. All three share close structural relationships with the Lrrn family and their overall genomic organisation, whereby the coding region is almost exclusively contained on a single exon. Members of the FLRT family are FGF-responsive, encode binding partners for FGF receptors (FGFRs) and are likely to function as feedback modulators of FGF-MAP kinase signaling [57, 58]. The LRRTMs form a four-member gene family widely expressed in the embryonic and adult mouse CNS [56, 59]. The archetypal member of the LINGO/LRRN6/LERN family, LINGO-I, is a component of the Nogo66 receptor/p75 signalling complex . The Lrrn and LINGO families share the same number of LRRs and each possesses a single Ig and FnIII domain. Although the embryonic expression of LINGOs in mouse has not been described in detail, chick LINGO-1 displays a number of overlapping and complementary sites of expression with Lrrn1 . Careful comparison between the expression patterns of the Lrrn, LRRTM and LINGO family members in the CNS of mouse and chick should point to possible functional similarities between them.
Dynamic expression of Lrrn1 in boundary regions
Lrrn1 is expressed throughout the neural plate of the chick embryo from its initiation, later becoming downregulated in a number of CNS regions that form boundaries. In the ventral midline, downregulation is coincident with floor plate formation following regression of Hensen's node from HH5/6 onwards. Similarly, in the dorsal midline (roof plate) downregulation is coincident with the onset of neural tube closure from HH6/7. The formation of both floor plate and roof plate is associated with the early differentiation of specialized cell types and the acquisition of signalling properties [2, 3]. Thus, the loss of Lrrn1 from midline regions appears to correlate closely with the appearance of boundary properties and loss of neuronal precursor phenotype in them. At other boundaries, for example, in the mid-hindbrain, a temporal correlation does not hold. In chick, the MHB becomes morphologically distinguishable from HH8–9 whereas Lrrn1 downregulation becomes evident from HH12 and covers a domain that is considerably larger than the MHB itself, encompassing the caudal region of the midbrain, isthmus and r1.
Lrrn1 exhibits a biphasic expression pattern at rhombomere boundaries. In the first phase it becomes downregulated at boundaries and in the second, its expression is reactivated in the boundaries themselves. Lrrn1 downregulation at inter-rhombomeric boundaries becomes evident from HH14, several stages after their morphological appearance at HH9/10. Thus, it is unlikely that down-regulation plays a specific role in the initiation or establishment of boundaries in the mid-hindbrain region. One of the earliest detectable features of hindbrain boundaries is the local reduction in the rate of inter-kinetic nuclear migration, detectable from HH10 . Furthermore, an increase in intercellular space in boundaries compared to non-boundary regions is also detectable from the same stage. The rate of increase remains constant until HH14, after which it becomes accelerated . It is possible that Lrrn1 down-regulation is involved in this process of cellular rearrangement, since this is the stage when down-regulation in the basal plate of the hindbrain becomes evident. Although some markers have been characterized that are activated in boundary regions specifically around this stage, such as the promyelocytic leukemia zinc finger gene (plzf), fgf3 and Pax6 [16, 61, 62], further work is required to pinpoint its relationship to other genes that are involved in such a process. Lrrn1 upregulation in rhombomere boundaries between HH17 and HH18 coincides with the emergence of a morphologically and genetically distinct boundary cell population [16, 18]. Thus, the dynamics of Lrrn1 expressions at boundaries may prove useful in understanding the cellular and genetic mechanisms involved in boundary maintenance and consolidation or the emergence of defined cell types with distinct boundary cell properties.
A similar pattern of downregulation followed by activation is also seen at the ZLI during a key stage of diencephalic development. The ZLI functions as a local organizer required for region-specific gene expression in the prethalamus and thalamus through the expression of Shh [8, 9]. It forms a narrow compartment flanked anteriorly and posteriorly by linage restriction boundaries that bisects the diencephalon transversely . The first indication of the position of the ZLI at earlier stages is the exclusion of Lfng from a wedge-shaped domain at HH12 . Lrrn1 is also downregulated in the dorsal region of the Lfng-free prZLI at the same time and in the future thalamus, but expression remains in the prZLI until HH15. Expression then clears from the prZLI immediately prior to the intrusion of Shh into the ZLI from the basal region of the diencephalon. The timing of Lrrn1 down-regulation suggests that it may be involved in regulating the competence of this area of the neuroepithelium to express Shh. From HH17 onwards, Lrrn1 flanks the Shh domain at its anterior margin and remains highly expressed in this region. During the preparation of this work, another study describing the expression of chick Lrrn1 in the CNS was published suggesting that Lrrn1 prefigures the position of the ZLI before its overt formation . Our results confirm and extend these findings by clearly showing, using double in situ hybridisation techniques, that Lrrn1 downregulation immediately precedes Shh expression and, thus, the emergence of organizer function.
Correlation with the function of TRN and CAPS
The functional roles of TRN and CAPS in cellular recognition have been best characterized in the fly wing imaginal disc. Here they also demonstrate a biphasic pattern of expression with apparently distinct and separable functions. Early expression is restricted to the D compartment of the wing disc during the second instar. Early function seems to be to specify D cell affinity properties, which are important for the establishment of the DV boundary, requiring both extracellular and intracellular portions of the proteins [30, 31]. During the third instar, expression becomes restricted to the lateral regions of the wing primordium and no longer respects the DV boundary. The later function for TRN/CAPS appears to be as cell survival factors and only requires the extracellular domain [31, 63]. We have gone to great lengths to obtain functional evidence of a role for Lrrn1 in boundary formation/maintenance using gain of function (over expression) and loss of function (shRNA-mediated knockdown) approaches via in ovo electroporation. Neither of these has been successful in generating a discernable phenotype. This is not without precedent for LRR-containing proteins – the function of TRN/CAPS as specifiers of D cell affinity were only revealed via gain-of-function in a mutant background where normal expression had been removed. Loss-of-function experiments using TRN/CAPS double mutant clones also did not have an overt effect on the patterning of the wing. Hence, it is possible that we will need to generate suitable loss-of-function constructs to interfere with Lrrn1 effectively.
Work from Stephen Cohen's group has predicted the existence of an as-yet uncharacterized cellular receptor for TRN/CAPS and found no evidence of homophilic interactions [30, 31, 63]. However, a recent study examining the function of the same proteins in the leg imaginal disc suggests that CAPS, and perhaps TRN also, act homophilically as cell-cell recognition molecules to mediate boundary refinement and induce invasive cellular behaviour . TRN/CAPS show limited but significant sequence similarity to chick Lrrn1 in their extracellular regions (26% across the LRRNT-LRR-LRRCT portions of the proteins, data not shown) and a short region of identity within the membrane proximal intracellular domain ('QKT' motif; Additional file 3). Although no evidence exists that points to TRN/CAPS and Lrrn1 being functional homologues, a hypomorphic mutant of CAPS has been identified that carries a point mutation (T501I), which is conserved between the three proteins and may prove useful in directing the design of loss-of-function tools for Lrrn1. Furthermore, TRN/CAPS also show a similar distribution of punctate cellular staining in the leg disc as we have seen for Lrrn1 in chick. We have adopted strategies to identify a ligand or co-receptor for Lrrn1 that have been widely employed to identify interacting partners for similar proteins. We have screened more than 5 × 105 clones of an embryonic mouse cDNA expression library  using the extracellular portion of Lrrn1 fused to alkaline phosphatase as a probe. We have also used the same reagent to look for binding to wholemount embryos and fresh-frozen cryostat sections under a wide range of binding conditions. Neither approach has proved successful. Furthermore, we do not see aggregation of Lrrn1 transfected cells in a manner that suggests homophilic cell adhesion, as has been seen for other LRR proteins such as CAPS  or FLRT3 . Thus, our evidence, albeit negative, strongly points to the fact that Lrrn1 does not interact homophilically. Nor does it appear to simply bind another ligand or receptor in trans. Thus, it is likely either to form a multiprotein receptor complex with other coreceptors or to bind to a complex or small molecule ligand.
The conserved nature of Lrrn1 suggests that it plays an important function during CNS development. It is dynamically expressed throughout the neuroepithelium but is restricted to NPCs and broadly downregulated in neuromere compartment boundaries. The endosomal localisation of Lrrn1 suggests that it may be involved in the regulation of signalling in progenitor cells. By analogy to related proteins, it has the potential to scaffold (via a PDZ-domain binding site) and regulate the subcellular trafficking of groups of functionally interacting molecules, such as receptors or ion channels, and could thereby regulate the integration of signalling pathways in NPCs.
Materials and methods
Cloning of chick Lrrn1and sequence analysis
Degenerate primers were designed to conserved amino acids of human (GenBank: AAQ88679), mouse (GenBank: NP_032542) and Xenopus (GenBank: AAH59292) Lrrn1. These corresponded to the peptide sequences CDCVIHW (amino acids 375–381: 5'-TGYGAYTGTGTMMTSCRYTGG-3') and PEPEIYW (amino acids 453–459: 5'-CCHSARCCMGARATHTACTGG-3'; Figure 1). Primers were used to amplify HH10–12 chick embryo cDNA (a gift of Dr D Chambers) with a 3' NotI-(dT)17 primer. The resulting 1,689 base-pair (bp) fragment encompassing the carboxy-terminal coding region (amino acids 453–717) and 3'-UTR (872 bp) of Lrrn1, was cloned into pCR4®TOPO® (Invitrogen. Paisley, Renfrewshire, UK) and verified by sequencing (pCR4TOPO-cLrrn1). A 575 bp SacI-SspI fragment of the Lrrn1 cDNA was then used to generate a radioactively labelled probe with [α-32P]dCTP. Screening a λZAPII cDNA library (gift of Prof. D Wilkinson) resulted in the identification of a 2.9 kb full-length clone, which was verified by DNA sequencing (GenBank accession number EF512462). Multiple sequence alignments were performed using ClustalW  using default parameters and annotated using ESPript . Protein motifs were identified using the InterProScan suite of programs  or Scansite .
Chick (Rhode Island Red) or mouse (CD11) embryos were fixed overnight at 4°C in 4% (w/v) paraformaldehyde (PFA) in phosphate buffered saline (PBS; pH7.2). Chick embryos were staged according to Hamburger and Hamilton (HH) . In situ hybridisation was performed as described previously . pCR4TOPO-cLrrn1 was linearized with PvuII and transcribed with T3 RNA polymerase (Roche, Burgess Hill, West Sussex, UK) to generate an antisense digoxigenin (DIG)-uracil triphosphate (UTP)-labelled riboprobe (DIG RNA Labeling Mix, Roche). A sense probe, transcribed with T7 RNA polymerase after linearization with SpeI, gave no specific signal (data not shown). For double in situ hybridisation, the second probe was labelled with fluorescein isothiocyanate (FITC)-dUTP (FITC RNA Labeling Mix, Roche) and both DIG/FITC probes (1 ng/ml each) were hybridized concurrently. After completion of the detection step for DIG with NBT/BCIP as a substrate (NBT/BCIP Stock Solution, Roche; 9.4 μg/ml nitroblue tetrazolium chloride and 4.7μg/ml 5-bromo-4-chloro-3-indolyl-phosphate), alkaline phosphatase (AP) conjugated sheep anti-DIG Fab antibody (Roche) was inactivated by washing embryos (3 × 5 minutes) in 0.1 M glycine-HCl (pH 2.2). The FITC detection step was then performed using sheep anti-FITC-AP (Roche) and Vector® Red substrate (Vector Laboratories, Burlingame, CA, USA). Additional plasmid templates were used to generate antisense riboprobes as follows: mouse Lrrn1 (HindIII-T7) ; chick Shh (SalI-SP6; gift of T Lints); chick Fgf8 (EcoR1-T7; gift of G Martin); chick lunatic fringe (ClaI-T3; gift of C Tabin).
Embryos were refixed overnight in PFA and cleared in 87% (v/v) glycerol prior to digital photography of wholemounts (Olympus DP70 attached to a stereo dissecting microscope). Some specimens were dissected and flatmounted under a No. 1.5 coverslip in glycerol. Vibratome (Leica VT 1000S) sections were cut at 40–50 μm after embedding tissue in 20% (w/v) gelatine/PBS. Following infiltration in gelatine at 65°C for 1–2 h, tissue was embedded and post-fixed in PFA containing 0.1% (w/v) glutaraldehyde. Flatmounts and sections were photographed on a Zeiss Axiophot microscope equipped with a Zeiss Axiocam or Olympus DP70 CCD camera.
Generation of anti-Lrrn1 antisera
A fragment corresponding to the intracellular domain of the chick Lrrn1 (IC, amino acids 660–716), including the translational termination codon, was generated by PCR with primers 5'-ACGGATCCAAAACTACCACCATTCAC-3' and 5'-CGCCTCGAGTTACCACATGTAATAGC-3', cloned into the pCR4®TOPO® vector (Invitrogen) and verified by sequencing. IC was fused in frame to GST) by cloning a BamHI-XhoI fragment (restriction sites underlined above) into pGEX-5X-3 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The GST-IC fusion protein was expressed in Escerichia coli (BL21 strain, Promega, Southampton, Hampshire, UK) and purified on a glutathione sepharose 4B column (Microspin, GE Healthcare) following lysis and mild sonication. Polyclonal antisera against the purified GST-IC protein were raised in rabbits (Eurogentec, Seraing, Belgium).
Western blotting and PNGase F treatment
Embryo or cell extracts were made by homgenization (via pipetting) and lysis in ice-cold RIPA buffer containing a cocktail of protease inhibitors (Complete, Roche). Lysates were extracted for 1–2 h at 4°C with gentle rocking followed by microcentrifugation (13,000 × g at 4°C for 20 minutes). Cleared lysates were stored at -20°C. Western blotting was performed with supported nitrocellulose membranes using standard techniques following denaturing SDS-PAGE on an 8% polyacrylamide gel (Mini-Protean III system. Bio-Rad, Hemel Hempstead, Hertfordshire, UK). The GST-IC antibody was used at a dilution of 1:6,000 and detected using a horseradish peroxidase-coupled secondary antibody (Vector Laboratories, 1:1,000) followed by an enhanced chemiluminescent detection system (Pierce, Rockford, IL, USA). PNGase F (New England Biolabs, Hitchin, Hertfordshire, UK) digestion was performed on 15 μl (approximately 20 μg) of HH18 embryo lysate or 5 μl (approximately 5 μg) of Lrrn1-GFP transfected Chinese hamster ovary (CHO) cell lysate according to the manufacturers instructions, using a total of 2 μl (1,000 U) of PNGase F (2 h at 37°C).
Unfixed chick embryos were directly embedded in OCT compound (Sakura Finetech, Zoeterwoude, (The Netherlands) on dry ice and stored at -80°C. Frozen sections (8–20 μm) were cut on a Bright OTF cryostat at -15°C and dried briefly (30 s) on a hotplate at 55°C and then fixed for 10 minutes at room temperature using PFA. Longer fixation protocols resulted in a dramatic reduction in signal:noise. Sections were washed in TBST (100 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v/v) TritonX-100), blocked with 10% (v/v) foetal calf serum (FCS) in TBST for 1 h at room temperature and incubated in primary antibody (in block) overnight at 4°C. Sections were washed in TBST and incubated in fluorescently conjugated goat secondary antibodies (Alexa Fluor®, Invitrogen) diluted 1:1–2,000 in TBST/1% (v/v) FCS for 1 h at room temperature. After washing in TBST, sections were coverslipped in a glycerol-based mounting medium (Citifluor, London, UK). Nuclei were conterstained with Hoechst 33342 (0.3μg/ml, Invitrogen) by inclusion in the final wash step. Sections were photographed on a Nikon Eclipse microscope using a Zeiss Axiocam.
Cell transfection and immunocytochemistry
Mouse neuroblastoma (Neuro2a) or human HeLa cells were seeded on sterile 13 mm diameter glass coverslips (No. 1.5) in DMEM supplemented with 10% (v/v) FCS, 2 mM L-Alanyl-L-Glutamine (GlutaMAX™I, Invitrogen), 1,000 u/ml penicillin, 1,000μg/ml streptomycin and 25μg/ml of amphotericin B (Antibiotic-Antimycotic, Invitrogen) in a humidified 37°C incubator/8% (v/v) CO2. Cells were transfected at 30–50% confluence with either a carboxy-terminal  or amino-terminal (gift of Dr Bryan Haines) FLAG-tagged mouse Lrrn1 expression vector. For live labelling experiments, N-FLAG-Lrrn1 expressing cells were incubated with mouse anti-FLAG® M2 monoclonal antibody (1:500, Sigma, Gillingham, Dorset, UK), or also in the presence of Alexa® Fluor 633-labelled EGF (10μg/ml, Invitrogen), for 30 minutes at 37°C. They were washed by transferring between 4 wells containing 500μl of warm culture media and fixed by adding 500μl 8% (w/v) PFA to the final wash step. Cells were washed in PBS and permeabilized with 0.3% (v/v) TritonX-100/PBS for 10 minutes. Total Lrrn1 protein was detected with the IC antibody (1:1,500) followed by an Alexa® Fluor 488 goat anti-rabbit secondary antibody (1:2,000, Invitrogen). The antibody against EEA1 (rabbit polyclonal antiserum 1:1,500) was a kind gift of Dr Mario Zerial (EMBL, Heidelberg, Germany) and the anti-human transferrin receptor (rabbit polyclonal, 1:250) was from Chemicon. Millipore, Watford, Herfordshire, UK) Internalized anti-FLAG was detected with an Alexa® Fluor 568 goat anti-mouse secondary antibody. Cells were mounted in Citifluor and imaged on a Zeiss LSM 510 confocal microscope using 63× (NA 1.3) oil immersion objective.
We would like to acknowledge: Rajeev Gupta, Bryan Haines, the late Dennis Summerbell and Peter Rigby for mouse Lrrn1 reagents and advice. Mario Zerial for the anti-EEA1 antisera. Members of the MRC Centre for Developmental Neurobiology and the Lumsden Lab for helpful discussions, in particular Dr Clemens Kiecker and Dr Stefen Scholpp for critical reading of the manuscript. The 5E1 monoclonal antibody developed by Prof. TM Jessell was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA.
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