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Pioneer Axon Guidance by UNC-129, a C. elegans TGF-β

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Science  31 Jul 1998:
Vol. 281, Issue 5377, pp. 706-709
DOI: 10.1126/science.281.5377.706

Abstract

The unc-129 gene, like the unc-6 netrin gene, is required to guide pioneer motoraxons along the dorsoventral axis ofCaenorhabditis elegans. unc-129 encodes a member of the transforming growth factor–β (TGF-β) superfamily of secreted signaling molecules and is expressed in dorsal, but not ventral, rows of body wall muscles. Ectopic expression of UNC-129 from ventral body wall muscle disrupts growth cone and cell migrations that normally occur along the dorsoventral axis. Thus, UNC-129 mediates expression of dorsoventral polarity information required for axon guidance and guided cell migrations in C. elegans.

Axon guidance along the dorsoventral (D/V) axis of animals of diverse phyla involves secreted, laminin-related, UNC-6/netrin guidance cues (1). The signaling pathways activated by these molecules require the UNC-5 and UNC-40/DCC transmembrane receptor families (2–4). In C. elegans, mutations inunc-129 (5) cause defects in the dorsally oriented trajectories of motoraxons that resemble those present inunc-5, unc-6, and unc-40 mutants (5, 6).

A 6.5-kb genomic subclone of cosmid C53D6 was able to rescue the uncoordinated phenotype of unc-129 mutants after germline transformation (7, 8) (Fig. 1A). Sequence analysis by the C. elegans genome-sequencing consortium (9) revealed a single open reading frame on this fragment that encodes a protein related to the TGF-β superfamily. The corresponding 1.5-kb cDNA (10) includes 5 exons, 34 base pairs (bp) of 5′ untranslated region (UTR), and 281 bp of 3′ UTR and is predicted to encode a protein of 407 amino acids (Fig. 1B). Northern (RNA) analysis of wild-type mRNA revealed a single transcript (11) consistent with the size of the cDNA. The 6.5-kb rescuing genomic fragment includes 3 kb of 5′ promoter sequence. A minigene containing 4.5 kb of 5′ promoter sequence fused to the unc-129 cDNA was able to rescue the phenotype of unc-129 mutants, indicating that there are no essential regulatory elements in introns or the 3′ sequence (12).

Figure 1

Positional cloning and primary sequence of unc-129. (A) Cosmids spanning the mes-6fem-3 interval were assayed forunc-129 rescuing activity (+ or −). The rescuing region was delimited by testing genomic subclones and an unc-129minigene. Genomic structure is indicated by boxed regions, black (coding), white (3′ UTR). Restriction sites: c, Cla I; x, Xba I; s, Sma I; b, Bst EII; bg, Bgl II; a, Ahd I; bm, Bsp MI. (B) Peptide sequence of UNC-129 with putative signal sequence (underlined), putative cleavage site (boxed), and conserved cysteines (highlighted in bold). Amino acid substitutions in unc-129alleles are indicated above the sequence (arrows). Asterisks indicate stop codons. (C) Comparison of the mature region of UNC-129 with members of the TGF-β superfamily; DPP, 60A (Drosophila); DAF-7 (C. elegans); BMP-2 (chicken); Nodal (mouse); BMP-7, MIS, GDF-1, TGF-β2 (human). The interdomain region predicted to be absent from UNC-129 is boxed (unc-129 is gene C53D6.2, accession number AF029887). Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

UNC-129 shares features with the TGF-β superfamily, including a signal sequence, a prodomain, and a COOH-terminal region that contains seven conserved cysteines (13). The UNC-129 COOH-terminal sequence identity ranges from 33% with human BMP-7 to 24% with TGF-β2. Thus, unc-129 likely represents a subfamily of the TGF-β superfamily.

Sequence analysis revealed the absence of residues in UNC-129 that would be expected between the α-helical region and β sheet of TGF-β molecules (Fig. 1C) (14). This interdomain region forms a β turn with a protruding loop accessible to solvent. The three-dimensional structures of TGF-β1 and TGF-β2 differ at this site, which may promote their differing receptor-binding affinities (15). Deletion of the loop in TGF-β1 abolishes certain TGF-β1–mediated responses (16). Without knowledge of the crystal structure of UNC-129, it remains unclear whether the missing residues form the COOH-terminal end of the long α-helix or affect receptor specificity.

In C. elegans, TGF-β signaling pathways have been implicated in control of dauer larva formation, male tail patterning, and body size (17–19). These functions appear unaffected in unc-129 null mutants. Rather,unc-129 mutations disrupt axon guidance. No other patterning or morphological defects were identified. ev557 andev554 mutations, which introduce stop codons (Fig. 1B) (20), cause axon guidance defects with higher penetrance than the hypomorph, ev566 (5). Mutations in known components of TGF-β signaling pathways did not cause any axon guidance defects in the DA (n = 120) or DB (n = 120) classes of motorneurons (21). Alleles tested weredaf-1(m213ts) anddaf-4(e1364ts), which are genes encoding type I and type II serine-threonine kinase receptors, respectively (17, 18).

We assessed the expression pattern of unc-129 usingunc-129::gfp transcriptional reporter genes (22) expressed from transgenic arrays (Fig. 2, A to E). Promoter activity was first detected at late gastrulation stage in cells that include descendants of the AB and E lineages. Expression continues through embryonic elongation in some of these cells. About 450 to 520 min after first cleavage, expression was observed in a subset of cells in the head, including one ventral muscle, and in all dorsal body wall muscles. Between 520 min and hatching, green fluorescent protein (GFP) expression was detected in the DA and DB classes of motorneurons, excluding DA8 and DA9. This pattern of expression persisted into the adult stage. In addition, expression was detected in a subset of cells in the head and in pharyngeal neurons and muscle. Of these, only interneuron I4 and muscle m8 were unambiguously identified. In late larval stages, expression was detected in the spermatheca, seam cells, CAN, PDE socket, and four cells that encircle the vulva. Among cells that express unc-129 promoter activity, only DA and DB motorneurons display morphological or axon guidance defects.

Figure 2

(left).Expression pattern and cell nonautonomous activity ofunc-129. (A) Comparison ofunc-129::gfp transcriptional reporters used in this study with unc-129 rescuing genomic DNA. (Bto E) unc-129::gfp expression pattern at ∼280 min (B), ∼430 min (C), ∼550 min (D) postcleavage, and in LI larvae (E). Expression in dorsal body wall muscle [arrowheads in (D) and (E)] is observed before DA and DB expression. GFP expression vectors used in (B) to (D) contain a nuclear localization signal. (F and G) unc129::gfpreporter derivatives expressed predominantly in DA and DB motorneurons (F) (arrow indicates dorsal nerve cord) and dorsal body wall muscle in L1 larvae (G). (H) unc-129 expression from the dorsal muscle–specific promoter is sufficient to completely rescue theunc-129 phenotype. Restriction sites as in Fig. 1. Bar, 10 μm.

Deletion analysis revealed portions of the unc-129 5′ regulatory region that promoted predominantly muscle- or motorneuron-specific GFP expression (22, 23) (Fig. 2, F and G). These truncated regulatory regions were used to express wild-type unc-129 coding sequence in either the dorsal muscle or the DA and DB motorneurons to test for its ability to rescue the unc-129 mutant phenotype. UNC-129 expression from the dorsal body wall muscle-specific promoter, but not from the motorneuron-specific promoter, rescued the uncoordinated movement and axon guidance defects of unc-129 mutants (23) (Fig. 2H). This result suggests that wild-type UNC-129 expressed by dorsal muscle acts cell nonautonomously to guide the circumferential migrations of pioneer axons.

The timing of unc-129::gfp expression also suggests thatunc-129 mediates axon guidance. unc-129 is expressed in dorsal muscle at the twofold stage of embryogenesis (450 to 520 min) when DA, DB, and DD motoraxons grow (480 to 515 min) toward the dorsal midline (24). unc-129 is also expressed in dorsal muscle postembryonically as VD motoraxons grow toward the dorsal midline.

To further investigate whether dorsal-specific expression ofunc-129 is important for its function, we expressed a functional hemagglutinin (HA)-tagged UNC-129 (25) in both dorsal and ventral rows of body wall muscles (verified by immunostaining) under the control of the muscle-specificmyo-3 (myosin) promoter (26). We examined axon morphologies of the DA and DB neurons in independent lines of ectopic UNC-129–expressing animals using the neuron-specific GFP fusion to the unc-129 promoter. Wild-type worms transgenic for myo-3::unc-129HA displayed uncoordinated locomotory defects and axon guidance defects in the DA and DB neurons that resembled the defects observed inunc-129 mutants (Fig. 3), but with lower penetrance for the DA than for the DB class (Fig. 4A).

Figure 3

(right). Ectopic expression of unc-129 in dorsal and ventral body wall muscle phenocopies the axon guidance defects of an unc-129 null mutant. DA and DB neurons are visualized in L4 stage animals through use of the neuronal unc-129::gfp reporter. Motoraxons are misrouted longitudinally at lateral positions (arrows). (A) Wild type; (B) unc-129(ev557); and (C) unc-129(+) animal carrying an integratedmyo-3::unc-129HA transgene. Arrowheads indicate row of lateral seam cells. Bar, 50 μm.

Figure 4

Ectopic expression of unc-129 in all body wall muscle causes axon guidance and cell migration defects. (A) DA and DB axon guidance defects in wild type, anunc-129 mutant, and three independent lines carrying integrated myo-3::unc-129HA transgenes. All strains are transgenic for the neuronalunc-129::gfp reporter. (B) Ventral guidance defects in the AVM and PVM neurons. Axons were scored as ventral guidance defective if AVM and PVM axons were misrouted longitudinally along the lateral hypodermis. All strains are transgenic for a mec-4::gfp reporter. (C) DTC dorsal migration defects in wild-type and unc-5 mutants expressing myo-3::unc-129HA transgenes. Themyo-3 vector strain carries an extrachromosomal array containing the myo-3 expression vector alone. Bars represent ±SD of a binomial distribution of the same sample size and observed mean.

Ectopic unc-129 expression also causes axon guidance and cell migration errors that are not found in unc-129 mutants. We assessed ventral axon guidance by examining AVM and PVM mechanosensory neurons using a mec-4::gfpreporter. In both wild-type and unc-129 mutants, these neurons are located at lateral positions and send a single process toward the ventral nerve cord. In allmyo-3::unc-129HA transgenic lines, however, many AVM and PVM axons are misrouted along longitudinal trajectories (Fig. 4B). Similar ventral guidance defects are also found inunc-6 and unc-40 mutants (6). Therefore, unc-129 expression from all body wall muscle perturbs guidance of axon growth cones in both dorsal and ventral directions. We also observed defects in the dorsalward migrations of the distal tip cells (DTCs), which are mesodermal cells that normally follow a U-shaped trajectory along the body wall (Fig. 4C). Similar DTC migration defects are present in unc-5,unc-6, and unc-40 mutants (6). Thus, spatially restricted expression ofunc-129 promotes normal axon guidance and DTC migration (27).

Any model of UNC-129 function must take into account its genetic interactions with the UNC-6/netrin pathway, particularly UNC-5. The identification of unc-129 mutations as suppressors of ectopic UNC-5–induced growth cone guidance is consistent with either a direct role in the UNC-6/netrin pathway or a role in a parallel pathway of related function. The nearly complete penetrance of axon guidance defects exhibited by unc-5 and unc-6 null mutants compared to the similar but milder defects in unc-129 null mutants (5) precludes the use of double-mutant analysis to distinguish between these possibilities.

UNC-129 may act directly as a guidance cue that provides polarity information to migrating growth cones, or indirectly, by inducing neighboring cells to form a guidance cue. In principle, UNC-129 could affect expression of components of UNC-5 signaling. However, UNC-129 does not affect transcription of unc-5, unc-6, orunc-40 as judged by examining expression ofunc-5::gfp (28),unc-40::gfp (3), andunc-6::HA (29) reporter genes in wild-type and unc-129(ev554) animals (30). Because none of the known TGF-β receptors (including DAF-1 and DAF-4) affect motoraxon guidance and TGF-β or Smad mutants do not show uncoordinated phenotypes (17–19), we suggest that UNC-129 uses an unconventional TGF-β–based mechanism to guide axons on the D/V axis in C. elegans. One possibility, suggested by the finding that TGF-β molecules can bind to TSP type I domains (31), is that UNC-129 acts directly on UNC-5 to enhance the ability of UNC-5 to mediate UNC-6–dependent growth cone repulsion (away from the ventral midline). Or, UNC-129 may function in a separate signaling pathway that mediates motoraxon attraction to the dorsal midline. The latter model predicts that motoraxons would be simultaneously repelled and attracted dorsally by opposite gradients of UNC-6 and UNC-129, respectively (17, 18). The identification and localization of additional UNC-129 signaling pathway components should help to distinguish between these models.

  • * To whom correspondence should be addressed. E-mail: culotti{at}mshri.on.ca

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