1 Wellcome CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
2 Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232-2175, USA
* Present address: University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA
Author for correspondence (e-mail: jaa{at}mole.bio.cam.ac.uk)
Accepted 4 November 2001
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SUMMARY |
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Key words: C. elegans, vab-7, Motoneurone identity, Axons
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INTRODUCTION |
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Three motoneurone subtypes, DA, DB and DD, are incorporated into the C. elegans ventral nerve cord (VNC) during embryonic development (Sulston, 1983; Sulston et al., 1983
). Five additional subclasses of postembryonic motoneurones (VA, VB, VC, AS, VD) are added in the first larval stage (Sulston and Horvitz, 1977
). These motoneurones are grouped on the basis of common morphological characteristics, neurotransmitter expression and presynaptic specificity (White et al., 1986
). HD transcription factors that regulate subsets of these traits have been identified. The UNC-30 HD protein functions in DD and VD motoneurones (D-class) where it promotes expression of GABA pathway components and is required for normal neuronal morphogenesis (Jin et al., 1994
). The LIM-HD protein, LIN-11, is expressed in postmitotic VC motoneurones and mediates VC motor axon fasiculation in the ventral nerve cord (VNC) (Hobert et al., 1998
). The UNC-4 HD protein is expressed in A-class motoneurones (DA, VA) to prevent the adoption of B-class (DB, VB) traits (Miller and Niemeyer, 1995
). In unc-4 mutants, A-class motoneurones are morphologically normal but express a B-type nicotinic acetylcholine receptor (nAChR) subunit, acr-5 (Winnier et al., 1999
). In addition, mutations that disrupt unc-4 function result in the miswiring of VA motoneurones with presynaptic inputs normally reserved for B-class motoneurones (White et al., 1992
). The dependence of these UNC-4 activities on physical interaction with the Groucho-like transcriptional co-repressor protein, UNC-37, indicates that UNC-4 is likely to function as a negative regulator of B-class genes (Pflugrad et al., 1997
; Winnier et al., 1999
).
We show that the C. elegans Even-skipped homologue, VAB-7, is expressed in DB class motoneurones where it functions as a negative regulator of A-class traits. In vab-7 mutants, ectopic UNC-4 in DB motoneurones results in the adoption of DA type axonal trajectory and repression of the B-class acr-5 gene. Ectopic expression experiments indicate that vab-7 may also promote expression of B-class genes through a parallel pathway that does not depend on unc-4 function. These findings indicate that the proper differentiation of DA and DB motoneurones depends on HD transcription factors that reciprocally repress DB and DA traits, respectively. A related scheme appears to have been used on a grand scale in the vertebrate spinal cord where a similar but more elaborate set of mutually repressive HD proteins specify separate neuronal progenitor domains (Briscoe et al., 2000; Muhr et al., 2001
). Involvement of Eve HD proteins in cross-repression of neuronal fates might be conserved, as Eve in Drosophila and mouse blocks expression of an alternative program of neuronal differentiation that would otherwise disrupt the function of the motoneurone circuit (Moran-Rivard et al., 2001
; Pierani et al., 2001
). This work points to the utility of exploiting an organism with a simple, well-defined nervous system and powerful genetics to uncover evolutionarily conserved mechanisms of neuronal differentiation in the metazoan nerve cord.
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MATERIALS AND METHODS |
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Plasmid constructs
To construct unc-3::vab-7, the vab-7 promoter was removed from the pJA17 vab-7-rescuing plasmid (Ahringer, 1996) by SacII and SphI digestion. A 4.14kb DNA fragment containing putative unc-3 promoter to the beginning of exon 2 was amplified by PCR from the pBP6-1 plasmid (Prasad et al., 1998
) using UNC-3PP4 (5'-AAACTGCAGCCGCGGATGCCTGCAGGTCGAC-3') and UNC-3PP2 (5'-TTTCTGCAGCATGCCAGACCTGAGTAAGGTATTC-3') primers, cut with SacII and SphI and cloned into pJA17/SacII/SphIto create plasmid pJA55. The UNC-3::VAB-7 protein product will contain the first 30 amino acids of UNC-3 upstream of VAB-7, and an extra glycine residue at the fusion site that was created to allow in-frame cloning. This unc-3::vab-7 construct rescues the forward movement defect of vab-7 mutants (not shown). The acr-5::gfplacZ plasmid pJA63 was constructed by subcloning a 4.2kb SphI fragment containing the acr-5 promoter (from pJR7) (Winnier et al., 1999
) into the gfp::lacZ expression plasmid pPD96.62.
Generation of transgenic lines
DNA microinjection experiments were performed as previously described (Mello and Fire, 1995). weEx43 was generated by injection of wild-type hermaphrodites with 100 µg/ml of pRF4 [rol-6(d)] (Mello et al., 1991
) and 30 µg/ml of pJA55 (unc-3::vab-7). The weIs10 transgene was made by integration of an extrachromosomal array generated by injection of 30 µg/ml pJA55 (unc-3::vab-7) and 100 µg/ml pMH86 (dpy-20(+); (Clark et al., 1995
) into a dpy-20(e1282) background. The weIs10 line was integrated as described by Mello and Fire (Mello and Fire, 1995
) and was out crossed three times before analysis. weEx52 (acr-5::gfplacZ + rol-6(d)) was generated by injection of 30 µg/ml pJA63 and 100 µg/ml pRF4. ß-Galactosidase staining was performed as previously described (Fire et al., 1990
).
Generation of anti-VAB-7
A fragment containing the entire vab-7-coding region was subcloned into the His tag vector pRSETB (Clontech) to create plasmid pJA18. Protein was purified on a nickel column after denaturation. A mouse was injected seven times over a period of 11 months with 15 µg of His-tagged VAB-7 in Freunds complete adjuvant, followed by intravenous injection of 10 µg protein in phosphate-buffered saline (PBS). The animal was sacrificed 4 days later and the spleen frozen. After fusion of spleen cells, one monoclonal line (2C4) that gave bright staining with no background was obtained.
Immunostaining of embryos, larvae and adults
Immunostaining experiments were performed as follows: embryos isolated by hypochlorite treatment were placed on a poly-lysine coated slide, squashed under a coverslip and frozen on dry ice for 10 minutes. After freezing, the coverslip was flicked off, and 100 µl of 5% formaldehyde in PBS placed on the sample for 20 minutes in a humid chamber. After incubation, the slides were immediately placed in 100% methanol for 4 minutes, in PBS with 0.2% Tween (PBST) for 4 minutes, blocked in 1% non-fat milk in PBST for 10 minutes, then placed in PBST for 10 minutes. Monoclonal anti-VAB-7 primary antibody (from cell culture supernatant) or anti-ß-galactosidase antibody (Cappel) was incubated overnight at 4°C; secondary antibodies (FITC anti-mouse or Texas Red Amersham) were incubated for 1-2 hours at room temperature. Samples were mounted using mowiol. For staining of larvae and adults, worms were washed four times in 15 ml distilled water in 15 ml centrifuge tubes before they were placed on poly-lysine-coated slides for adhesion, and then treated as above.
Identification of neurones in the VNC
Identities of neurones in the ventral cord were assigned based on the position of their nuclei in the VNC, their commissures and their axonal processes in the DNC (Sulston, 1983; Sulston et al., 1983
; White et al., 1986
). Identification of postembryonic neurones expressing VAB-7 was aided by the unc-129::gfp marker, which is expressed in DA and DB motoneurones (Colavita et al., 1998
). Non-DB motoneurones were identified by comparing DAPI and unc-129::gfp staining data with that of White et al. (White et al., 1986
). For identification of neurones with ectopic ACR-5::GFPLACZ in weIs10;weEx52 (acr-5::gfplacZ) animals, the unc-17::gfp neuronal marker which is expressed in DA, DB, AS, VA, VB and VC motoneurones in the VNC was used (Lickteig et al., 2001
) (Rand et al., 2000
).
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RESULTS |
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To discover whether VAB-7 is expressed in the nervous system, a mouse monoclonal antibody was generated against VAB-7 recombinant protein. Staining embryos with this anti-VAB-7 antibody confirmed that VAB-7 is expressed in posterior muscle and epidermal cells (Fig. 1A,B). However, we also discovered a second phase of VAB-7 expression in embryonic ventral nerve cord (VNC) neurones, beginning at the 1.5-fold stage (Fig. 1 C). At the threefold stage VAB-7 is expressed in nine neuronal nuclei (seven in the VNC and two nuclei in the head), and in the five most posterior epidermal nuclei, which form the hyp8 to hyp11 cells (Fig. 1C-E). The vab-7(e1562) allele [which introduces an early stop codon (Ahringer, 1996)] appears to be null, as VAB-7 is not detectable in these embryos (data not shown).
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DB motoneurones exhibit reversed axonal polarity in vab-7 mutants
To determine if the lack of vab-7 expression perturbs the generation of DB motoneurones, we viewed DB neurones using unc-129::gfp, a promoter fusion that drives GFP expression in DA and DB cell bodies and processes (Colavita et al., 1998). We found that all DB neurones are generated in vab-7 mutants (compare Fig. 2B with 2C), indicating that vab-7 is not required for the production of these cells. In addition, two other DB characteristics are normal in vab-7 mutants. First, each DB commissure (White et al., 1986
) travels around the body on the correct side (i.e. right or left) to reach the dorsal nerve cord (data not shown). Second, as in wild-type, DB neurones express unc-17/cha-1::gfp (see Fig. 6B,D and data not shown), a marker for acetylcholine production (Lickteig et al., 2001
) (Rand et al., 2000
).
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UNC-4 function depends on physical interaction with UNC-37, a ubiquitously expressed Groucho-like transcriptional co-repressor (Miller et al., 1993; Pflugrad et al., 1997
). Furthermore, the UNC-4/UNC-37 complex is known to function as a negative regulator of DB and VB motoneurone-specific genes (Winnier et al., 1999
). We used the missense allele, unc-37(e262), to determine whether the UNC-4-induced reversal of DB axonal polarity also requires wild-type UNC-37 activity. As shown Fig. 2G, DB axonal polarity is restored to its normal posterior trajectory in unc-37(e262); vab-7(e1562) mutant animals (Table 1). This finding indicates that the UNC-4 is likely to function as a negative regulator of DB genes that direct posterior axonal outgrowth and that this repression is sufficient to impose an anterior trajectory in vab-7 mutant animals.
vab-7 is required for acr-5 expression in DB motoneurones
acr-5 encodes an acetylcholine receptor subunit that is normally expressed in B-class (DB, VB) but not in A-class (DA, VA) motoneurones (Winnier et al., 1999) (Fig. 4A). In vab-7(e1562) mutants, however, we found that acr-5::gfp expression is specifically lost from the DBs (Fig. 4B; Table 4). It has previously been shown that acr-5::gfp is negatively regulated by UNC-4 and its co-factor UNC-37 in DA and VA motoneurones (Winnier et al., 1999
) (Fig. 4C). Given that unc-4 is derepressed in the DBs of vab-7 mutants (Fig. 3B), loss of acr-5::gfp expression could be due to the ectopic expression of unc-4. This is indeed the case, as acr-5::gfp expression is restored to the DBs in unc-4; vab-7 double mutants (Fig. 4D; Table 4). Repression of acr-5::gfp by ectopic UNC-4 also depends on unc-37 as DB motoneurones express acr-5::gfp in unc-37; vab-7 animals (Table 4). Therefore, in DB motoneurones, vab-7 effectively promotes acr-5::gfp expression by repressing UNC-4 repressor activity.
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Animals carrying the unc-3::vab-7 transgene weIs10 can move forwards, but exhibit a strong backwards movement defect (data not shown). This striking UNC-4-like phenotype indicates that A-type motoneurones (DA and VA) may be affected. If the ectopically expressed VAB-7 protein retains wild-type function, then unc-4 should be repressed in the DA and VA motoneurones. This is indeed the case: unc-4::lacZ expression is lost from A-type motoneurones in the unc-3::vab-7 background (Fig. 3D; data not shown). Furthermore, we also found that acr-5::gfp is now ectopically expressed in the DAs and VAs in this transgenic line as would be expected from the inhibition of unc-4 activity in these cells by ectopic VAB-7 (Fig. 4E; Table 5).
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vab-7 function is necessary for fasiculation of dorsal as well as ventral nerve cords
In addition to the axonal polarity reversals of DB motoneurones, vab-7(e1562) mutants also show significant disorganization of neuronal processes in both the dorsal and ventral nerve cords (Fig. 6B,D). Although the polarity reversal of vab-7 mutants is rescued by removal of unc-4 activity, this defasiculation defect remains (Fig. 2F), indicating that abnormal polarity is not the cause of defasiculation. In addition, abnormal expression of unc-4 in DB motoneurones does not account for the disrupted fasicular organization of the axial nerve cords. We conclude that vab-7 must mediate some other DB-trait that in turn is necessary for proper process placement in both the dorsal and ventral nerve cords. To test this idea, we examined nerve cord fasiculation in vab-7 mutants ectopically expressing VAB-7 from the unc-3::vab-7 transgene. We found that the unc-3::vab-7 transgene rescues both the fasiculation (Fig. 6E) and forward movement defects of vab-7 mutants (data not shown). Interestingly, although the DNCs and VNCs of vab-7 mutants appear defasiculated when viewed using the unc-17::gfp reporter (expressed in all cholinergic neurones; Fig. 6B,D), fasiculation appears normal in the VNC but not the DNC when viewed with unc-129::gfp, a reporter expressed only in DA and DB neurones (Fig. 6F). This suggests that neurones other than DAs and DBs are defasiculated in the VNC. Our results indicate that DB neurones might have an important role in bundling in the nerve cords.
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DISCUSSION |
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The DB motoneurone fate
VAB-7 appears to have two roles in DB fate determination. First, VAB-7 blocks expression of the A-class gene, unc-4, in DBs (Fig. 7). In vab-7 mutants, ectopic expression of UNC-4 represses B-class genes and induces DB motoneurones to adopt the anteriorly directed axonal trajectory of DA motoneurones. Second, VAB-7 promotes DB characteristics independently of UNC-4 repression. Ectopic expression of VAB-7 in cholinergic motoneurone classes that do not express UNC-4 is sufficient to induce expression of the B-class genes (i.e. acr-5, unc-129) and to impose the posterior polarity characteristic of DB motor axons. Posterior DB polarity also appears to be controlled by another, as yet unknown pathway, as this trait is normal in unc-4; vab-7 double mutants. Finally, as discussed below, the defasciculation defects observed in unc-4; vab-7 animals reveal an independent vab-7 function that is necessary for proper bundling of processes in both the dorsal and ventral nerve cords.
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Fasciculation and eve in Drosophila and C. elegans
In vab-7 mutants, both dorsal and ventral nerve cords are defasciculated. This defect is not rescued by restoring proper posterior polarity of DB neurones (by removing unc-4), but is rescued by ectopic VAB-7 expression, suggesting that vab-7, and possibly DB neurones promote process bundling. Interestingly, Even-skipped in Drosophila also has a role in fasciculation (Landgraf et al., 1999). Axonal growth of Eve-expressing neurones (aCC and RP2) in the ISN nerve trunk and their subsequent innervation of dorsal muscles is dependent on Even-skipped. Furthermore, ectopic expression of Even-skipped in the nervous system promotes SN and ISN nerve trunk fasciculation. Landgraf et al. (Landgraf et al., 1999
) have provided indirect evidence that eve activity is required for expression of an unknown neuronal adhesion molecule. Mutations in a number of genes are known to cause nerve bundle defasciculation in C. elegans (McIntire et al., 1992
; Wightman et al., 1997
; Bloom and Horvitz, 1997
). One candidate for a downstream target of vab-7 is the
-integrin INA-1, which is expressed in DB (and other) neurones and is required for nerve bundle fasciculation (Baum and Garriga, 1997
).
Conservation of Even-skipped function
Genetic studies in C. elegans, Drosophila, and the mouse have shown that Even-skipped homologues function to distinguish alternative fates in the motoneurone circuit. In each case, Eve prevents one class of neurone from adopting traits normally reserved for another. In Drosophila, Eve is expressed in motoneurones that project along the ISN nerve to innervate dorsal muscles. In eve mutants, these motoneurones adopt the axonal trajectory of a different class of ISN motoneurones that synapse onto ventral muscles (Landgraf et al., 1999). Similarly, in vab-7 mutants in C. elegans, DB motoneurones reverse their normal posterior axonal polarity and instead assume the anteriorly directed trajectory of DA motor axons (this work). In the spinal cord of mouse Eux1 mutants, V0 interneurones are apparently transformed into V1 interneurones (Moran-Rivard et al., 2001
). At least in C. elegans and in Drosophila, ectopic expression of Even-skipped is also sufficient to impose axonal trajectories normally associated with eve-expressing motoneurones (Landgraf et al., 1999
) (this work).
A common element of Eve function in all three species is the repression of a downstream HD protein, which is normally expressed in the alternative neurone. In C. elegans, vab-7 prevents expression of the DA gene, unc-4, in DB motoneurones. Evx1 functions in mouse V0 neurones as a negative regulator of the engrailed homologue, En1, a marker for V1 cells (Saueressig et al., 1999; Moran-Rivard et al., 2001
). In Drosophila, ectopic expression of Eve is sufficient to inhibit Islet in ISN motoneurones (Landgraf et al., 1999
).
In addition to these similarities in Eve function, our work shows that VAB-7 functions within a reciprocally inhibitory network: VAB-7 inhibits the DA fate and UNC-4 inhibits the DB fate. Thus, one way that VAB-7 promotes DB differentiation is by blocking expression of a HD transcription factor that antagonizes DB traits. By extension, we propose that HD transcription factors in Drosophila and mouse are likely to antagonize fates promoted by Eve. For example, EN1 might exert a negative effect on V0 interneurone differentiation when ectopically expressed in V1 cells in Evx1 mutants just as UNC-4 inhibits DB fates in vab-7 mutant animals. In this case, normal V0 cell migration and axonal trajectory might be restored in Evx1 En1 double mutant mice (Saueressig et al., 1999; Moran-Rivard et al., 2001
). Both UNC-4 and EN1 include EH-1 domains that have been shown to recruit the transcriptional co-repressor protein, Groucho (Jimenez et al., 1997
; Winnier et al., 1999
). In the case of UNC-4, interactions with the nematode Groucho homologue, UNC-37, repress B-class motoneurone traits (Pflugrad et al., 1997
; Winnier et al., 1999
). Reciprocal inhibition by EH-1-containing HD proteins that recruit Groucho might be common, as recent work has revealed that such a mechanism in the vertebrate spinal cord defines distinct domains of neural progenitor cells (Muhr et al., 2001
). Thus, our work demonstrates that important elements of both the logic and molecular mechanisms employed by HD proteins in the specification of neuronal fates in the motor circuit have been preserved in evolution from nematodes to mammals.
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ACKNOWLEDGMENTS |
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