Netrin-3, a mouse homolog of human NTN2L, is highly expressed in sensory ganglia and shows differential binding to netrin receptors

Abstract

The netrins comprise a small phylogenetically conserved family of guidance cues important for guiding particular axonal growth cones to their targets. Two netrin genes, netrin-1 and netrin-2, have been described in chicken, but in mouse so far a single netrin gene, an ortholog of chick netrin-1, has been reported. We report the identification of a second mouse netrin gene, which we name netrin-3. Netrin-3 does not appear to be the ortholog of chick netrin-2 but is the ortholog of a recently identified human netrin gene termed NTN2L ("netrin-2-like"), as evidenced by a high degree of sequence conservation and by chromosomal localization. Netrin-3 is expressed in sensory ganglia, mesenchymal cells, and muscles during the time of peripheral nerve development but is largely excluded from the CNS at early stages of its development. The murine netrin-3 protein binds to netrin receptors of the DCC (deleted in colorectal cancer) family [DCC and neogenin] and the UNC5 family (UNC5H1, UNC5H2 and UNC5H3). Unlike chick netrin-1, however, murine netrin-3 binds to DCC with lower affinity than to the other four receptors. Consistent with this finding, although murine netrin-3 can mimic the outgrowth-promoting activity of netrin-1 on commissural axons, it has lower specific activity than netrin-1. Thus, like netrin-1, netrin-3 may also function in axon guidance during development but may function predominantly in the development of the peripheral nervous system and may act primarily through netrin receptors other than DCC.

Fig. 1.

Alignment of the predicted amino acid sequences of murine netrin-3, human NTN2L, chick netrin-2, and chick netrin-1 proteins. Amino acid residues identical among other netrins and murine netrin-3 are shaded in black. Similar residues are shaded in gray. The different domains of mouse netrin-3 (domains VI, V-1, V-2,V-3, and C) are indicated as defined by homology with other netrins (Serafini et al., 1994; Harris et al., 1996; Mitchell et al., 1996; de la Torre et al., 1997). The nucleotide sequence of mouse netrin-3 has been submitted to GenBank.

Fig. 2.

Ntn3 maps in the proximal region of mouse chromosome 17. Ntn3 was placed on chromosome 17 by interspecific backcross analysis. The segregation patterns ofNtn3 and flanking genes in 130 backcross animals that were typed for all loci are shown at the top. For individual pairs of loci, >130 animals were typed (see Materials and Methods). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus) F1 parent. Theblack boxes represent the presence of a C57BL/6J allele, and the white boxes represent the presence of aM. spretus allele. The number of offspring inheriting each type of chromosomes is listed at the bottom of eachcolumn. A partial chromosome 17 linkage map showing the location of Ntn3 in relation to linked genes is shown at the bottom. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the position of the loci in human chromosomes, where known, are shown to the right. References for the human map position of loci cited in this study can be obtained from Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of the Johns Hopkins University (Baltimore, MD).

Fig. 3.

Northern analysis of murinenetrin-3 expression in mRNA extracted from E9.5, E10.5, E11.5, and E12.5 whole embryos and E14.5, E16.5, and E18.5 neonatal and adult brain. The position of RNA molecular weight markers on the gel is indicated on the right. Elongation factor 1 a (EF1a) was used as a control for loading of mRNA.

Fig. 4.

Comparison of expression of murinenetrin-1 (A, E,I), murine netrin-3(B, F, J), chicknetrin-1 (C, G,K), and chick netrin-2(D, H, L) in the spinal cord by in situ hybridization. Comparable developmental stages in mouse and chick are illustrated: A–D, E9.5 in mouse (A, B) and stage 18 in chick (C, D); E–H, E10.5 in mouse (E, F) and stage 22 in chick (G, H); I–L, E11.5 in mouse (I, J) and stage 26 in chick (K, L). (Note that the signal along the edge of the ventricular zone and in the central canal inI and J represents an edge effect and not a real signal). sc, Spinal cord; d, dorsal root ganglion; fp, floor plate;vz, ventricular zone; dm, dermomyotome;s, somite. Scale bar, 0.25 mm.

Fig. 5.

Expression of murine netrin-3 and murine netrin-1 at different stages at the forelimb level. A–C, Murine netrin-3 at E10.5 (A), E11.5 (B), and E12.5 (C). D–F, Expression of murinenetrin-3 at E14.5 in transverse sections (D), with higher magnification showing expression in the lung (E) and in the muscles (E, F). G–I, Expression of murine netrin-1 at E14.5 in sections adjacent to those in D–F, respectively. Note that the expression patterns of netrin-1 andnetrin-3 are similar in muscles and the lung.d, Dorsal root ganglion; lb, limb bud;o, oesophagus; cm, condensing mesenchyme;m, muscle; l, lung; sc, spinal cord. Scale bar: A–D, G, 1.4 mm;E, F, H, I, 0.33 mm.

Fig. 6.

Expression of murine netrin-3 in the head region of the mouse embryo at E9.5 (A), E10.5 (B), E11.5 (C), and E12.5 (D). V, Trigeminal ganglion;VII, geniculate ganglion; VIII, vestibular ganglion; IX, glossopharyngeal ganglion;f, forebrain; h, hindbrain;m, mesenchyme. Arrows, Thalamus;arrowheads, positive signal in the ventral aspect of the neural tube in the hindbrain. Scale bar, 1.4 mm.

Fig. 7.

Murine netrin-3 protein binds to the netrin receptors DCC (A), neogenin (B), UNC5H1 (D), UNC5H2 (E), and UNC5H3 (F) but not a control protein of the immunoglobulin gene superfamily, L1 (C). Scale bar, 40 μm.

Fig. 8.

Equilibrium binding of the netrin-3(VI.V)-Fc fusion protein to UNC5H1, UNC5H2, UNC5H3, neogenin, and DCC. Binding of netrin-3(VI.V)-Fc was determined by measuring the radioactivity associated with cells after subsequent incubation with radiolabeled anti-human IgG antibody. Specific binding curves were fitted using the Hill equation. K_d values for the interaction of netrin-3(VI.V)-Fc with UNC5H1, UNC5H2, UNC5H3, neogenin, and DCC are 6.2, 3.3, 4.5, 3.0, and 11.5 nm, respectively.

Fig. 9.

Outgrowth-promoting and chemorepulsive actions of netrin-3 in vitro.A–C, E13 dorsal spinal cord explants were cocultured with 293 EBNA cells mock-transfected with the vector pCEP4 (A), with transfected 293 EBNA cells expressing netrin-3 (B) or with purified netrin-3 protein (120 μg/ml) (C). Netrin-3 promoted the outgrowth of spinal commissural axons from these explants. D,E, Explants of the ventral half of the HMJ of E11 rat embryos were cultured with 293 EBNA cells mock-transfected with the vector pCEP4 (D) or with transfected 293 EBNA cells expressing netrin-3 (E). Trochlear motor axons (arrows) originating from cell bodies in the trochlear nucleus (IV) extended dorsally away from the floor plate (FP) and into the collagen matrix. They continued to grow dorsally in the presence of control cells (D) but were repelled by netrin-3-secreting cells (E). In D and E,dots show the outline of aggregates of transfected cells. Scale bar, 33 μm.