unc-3, a gene required for axonal guidance in Caenorhabditis elegans,

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1 Development 125, (1998) Printed in Great Britain The Company of Biologists Limited 1998 DEV unc-3, a gene required for axonal guidance in Caenorhabditis elegans, encodes a member of the O/E family of transcription factors Brinda C. Prasad 1, Bing Ye 2, Randa Zackhary 2, Karen Schrader 1,3, Geraldine Seydoux 1 and Randall R. Reed 1,2,3, * 1 Department of Molecular Biology and Genetics, 2 Department of Neuroscience, and 3 Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA Author for correspondence ( rreed@jhmi.edu) Accepted 3 February; published on WWW 18 March 1998 SUMMARY The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons, as well as defects in dauer formation, a process requiring chemosensory inputs. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and/or neuronal differentiation in both sensory and motor neurons. Key words: Caenorhabditis elegans, Transcription, unc-3, Axonal pathfinding INTRODUCTION The central nervous system in mammals contains several classes of neurons, each with specialized function and morphology. The differentiative process that leads to the formation of this wide array of neuronal types is still poorly understood. In olfactory neurons, expression of specialized signal transduction components is thought to be regulated by the O/E family of transcription factors. The O/E transcription factors that have been identified (O/E-1, O/E-2, O/E-3) are expressed in olfactory neurons and their progenitors throughout development (Wang et al., 1997, Wang and Reed, 1993), and are thought to contribute to the terminal phenotype of these sensory neurons. Binding sites for these proteins are found upstream of several genes encoding components of the odorant signaling pathway. O/E proteins are also expressed transiently in a subset of neural precursor cells in the central and peripheral nervous systems (Davis and Reed, 1996). In addition, one member of the O/E family, O/E-1, is also expressed during B cell maturation, where downstream targets of the transcription factor have been identified (Hagman et al., 1993). Elucidation of the specific roles for members of this gene family in neural development, however, has been complicated by their overlapping patterns of expression (Wang et al., 1997). An O/E homologue, collier, has been identified in Drosophila (Crozatier et al., 1996). The collier mrna is detected in cells of the central and peripheral nervous systems of the developing embryo. The absence of mutations in the collier gene have prevented further insight into the role of this O/E family protein. The nematode C. elegans has proven a powerful system to study the function of proteins with specific roles in the development and activity of the nervous system. The observation that gene families represented by many members with overlapping functions in mammals are often represented by fewer members with unique functions in C. elegans led us to look for O/E homologues in this organism. In addition, the well-characterized cell lineages in the nematode allows for better understanding of the function of genes involved in the patterning of the nervous system. The C. elegans nervous system consists of 302 neurons of 118 types that connect in a reproducible manner (White et al., 1986; Ruvkun, 1997). The neurons are generated by invariant patterns of cell divisions and migrations (Sulston et al., 1983). Many of the neural types are generated by neuroblast sublineages that are reiterated along the symmetry axes of the worm. The ventral nerve cord is a large collection of axons that runs along the ventral midline of the body wall (White et al., 1975). The axons reach their synaptic targets with minimal branching. Most synapses occur en passant between axons running in parallel within the ventral cord (White et al., 1986). The absence of some neurons, miswiring of axons and defects in

2 1562 B. C. Prasad and others the surrounding hypodermis results in worms that are unable to move normally. We describe here the isolation of a C. elegans O/E protein and demonstrate that it corresponds to the previously characterized unc-3 locus. The uncoordinated (unc) loci define genes required in C. elegans for wild-type locomotion (Brenner, 1974). Genetic and phenotypic analysis of unc-3 mutants have demonstrated that this gene is required for proper differentiation of ventral cord motor neurons. Electron microscopic reconstructions of the ventral cord of unc-3 mutants revealed that the VA and VB classes of motor neurons have highly disorganized processes, neuromuscular junctions at ectopic sites and receive inappropriate synaptic input from interneurons due to the defasiculated axons (J. White, personal communication). In addition, mosaic analyses showed that unc-3 is likely to function cell autonomously in the ventral cord motor neurons, since loss of unc-3(+) activity in the lineages giving rise to motor neurons causes an Unc-3 phenotype (Herman, 1987). The defects observed in the ventral cord in unc-3 (e151) mutants were reminiscent of those seen after laser ablation of the neuron (AVG) that serves as the pioneering axon in the right bundle (Durbin, 1987) and indeed, the Unc-3 phenotype appeared more severe than the AVG-ablated animals with both defasiculation and locomotion defects. Recently, the defasiculation and miswiring defects of unc-3 mutants have been attributed to a primary requirement for unc-3 activity in the neurons that pioneer the left ventral cord during embryonic development (Wightman et al., 1997). Like its mammalian homologues, we find that unc-3 is expressed in motor neurons during axonogenesis, and is also expressed in one pair of chemosensory neurons, the ASI amphid neurons, throughout development. In addition to locomotory defects, unc-3 mutants also exhibit defects in dauer formation, suggesting that this protein regulates the differentiation of both motor neurons and sensory neurons in C. elegans. MATERIALS AND METHODS Identification and characterization CeO/E cdna and genomic clones A pair of degenerate oligonucleotides (AARTGYAAYCARAAYTG and TGRAANCKNCKCATRTC) based on sequences within exon 6 of the mammalian O/E genes was used in a PCR reaction (45 C annealing, 72 C extension, 94 C denaturation for 35 cycles) with 100 ng C. elegans genomic DNA as template. A product of the expected size (59 base pairs) was cloned into Bluescript KS and sequenced. This fragment was used to screen an EMBL3 C. elegans genomic DNA library (J. Nathans, JHU), and a single positive clone (λcog-3) was isolated and analyzed. A cosmid, F42D1, was subsequently identified in a BLAST homology search of the Sanger Center C. elegans genomic sequence database. The EMBL3 phage clone extended 4.2 kb into a cosmid gap at the left end of F42D1 (Fig. 1). cdna clones encoding CeO/E were identified by screening a λgt11 mixed stage cdna library (Pete Okkema, University of Illinois at Chicago) with a 32 P-labeled, 35-base pair oligonucleotide based on the DNA sequence of the cloned PCR product. The longest cdna clone obtained lacks an initiation methionine and encodes a protein 25 amino acids shorter at the amino terminus than the mammalian O/E proteins, suggesting that this cdna is not full-length. The 5 - most sequences of the CeO/E cdna were obtained by RT-PCR on first strand cdna generated from N2 worms using a specific antisense oligonucleotide in the coding region (nucleotides ) and a second oligonucleotide based on the SL1 splice leader sequence. The complete cdna and ORF of CeO/E were reconstructed from these fragments. Rescue of unc-3 phenotype A 12 kb SalI fragment derived from λcog-3 containing the entire CeO/E open reading frame was cloned into pbk-cmv (Stratagene). This plasmid was injected into unc-3(e151) hermaphrodites and F 2 progeny displaying wild-type movement were derived from three injected worms. In two of three independent rescued lines, the non- Unc worms expressed GFP from a coinjected marker, demonstrating that the phenotypically rescued worms received the injected DNA. Analysis of unc-3 mutants Exons 2 through 11 were PCR amplified from genomic DNA isolated from unc-3 mutants and sequenced using gene-specific primers. The PCR reactions were performed with Pfu polymerase and each allele was independently amplified with two different pairs of primers. The strains unc-3(e151) and unc-3(p1001) were obtained from the C. elegans Genetic Center, and unc-3(e54) was provided by C. Bargmann (UCSF). The e151 and e54 alleles were isolated at the MRC Laboratory of Molecular Biology and the p1001 allele was isolated in Dick Russell s laboratory. The e151 is a presumptive null allele since the phenotype of unc-3 (e151) homozygotes is similar to that of unc- 3 (e151)/df (Hodgkin, 1997). Analysis of expression patterns in embryos and adults An oligonucleotide encoding amino acid residues of the CeO/E protein and a BamHI restriction site was used in combination with an oligonucleotide from the vector to amplify a 4.2 kb region containing putative promoter and transcribed sequences up to the second exon of unc-3. The resulting fragment was cleaved with SalI and BamHI and cloned into ppd95.69, a plasmid containing GFP (A. Fire, J. Ahnn, EMBL3 λ clone Rescuing Fragment F42D1 1kb Sal1 ATG Eco RI EcoRV EcoRV Fragment used for GFP fusion Sal1 Sal1 Bgl II XbaI Xba I Stu I Bam HI Fig. 1. Schematic diagram of CeO/E genomic organization. Exons present in the cdna are indicated by filled boxes. The 6th exon, containing sequences used to identify full-length cdna and genomic clones, is shaded. Sequences present in the F42D1 cosmid, the 12 kb SalI unc-3 rescuing fragment and the EMBL3 genomic clone are indicated by lines above the genomic map. Sequences used in the unc-3::gfp fusion are indicated below the genomic map.

3 unc-3 encodes an O/E transcription factor 1563 S. Xu and G. Seydoux, unpublished data). This construct (pbp6-1) was coinjected with a plasmid encoding the dominant marker rol-6 (Mello et al., 1991) into N2 hermaphrodites, and roller lines that had incorporated the GFP reporter and selectable marker into extrachromosomal arrays were isolated. Four independent lines were isolated and one line was gamma-ray irradiated (Harald Hutter, JHU). The resulting strains rhex11 contained an extrachromosomal array and rhis11 contained a chromosomally integrated version of the array. These lines allowed us to study the morphology of the cell bodies and axons of the CeO/E-expressing cells. Mixed population of worms expressing unc-3::gfp were incubated with 0.2 µg/ml DiI in M9 for 2 hours. The worms were washed and transferred to a seeded plate for 2 hours, mounted on agar pads, and examined for GFP and DiI fluorescence. The location of DiI-labeled cells in relation to GFP-positive cells in the head was consistent with the identification of the GFP-positive chemosensory neurons as ASI. Generation of antibodies A synthetic C-terminal peptide (GAVNPFAATLQSSSRLS) was crosslinked at a 1:1 weight ratio to bovine serum albumin and 250 µg subcutaneously injected into rabbits. Immunoblotting with crude antisera after the second boost revealed a strong reaction to recombinant CeO/E protein expressed in a mammalian cell line. Immune serum was affinity purified by acid elution from a peptideconjugated column and stored at 80 C in 1%BSA/TBS (Davis and Reed, 1996). Embryos or mixed population of worms were freezecracked in PBS, fixed in methanol (15 minutes) and acetone (10 minutes) (Miller and Shakes, 1995), and stained with anti-ceo/e affinity-purified antibody (1:1000) and FITC-coupled or CY3-coupled goat anti-rabbit antibody (1:50 FITC and 1:1000 CY3). The secondary antibodies were preadsorbed with BSA to reduce non-specific staining (Miller and Shakes, 1995). To confirm that the anti-ceo/e antibody was specific for CeO/E, animals carrying unc-3 mutations were stained. No staining was observed in unc-3(e151) and unc-3(e54) mutants; in contrast, wildtype levels of staining were observed in unc-3(p1001). These results are consistent with the observation that unc-3(e151) and unc-3(e54) code for premature stop codons, whereas unc-3(p1001) codes for a missense mutation. Staining was also eliminated by blocking the anti-ceo/e antibody with 100 µg/ml of peptide to which the antibody was raised. To confirm that all DA neurons express CeO/E, we stained embryos expressing an integrated 3.4 kb PstI-XmaI unc-4 promoter::gfp fusion (line rhis7, Harald Hutter, JHU) with anti-ceo/e antibodies. In embryos, the unc-4::gfp fusion is expressed in the DA neurons in the ventral cord and in the SAB ring interneurons in the nerve ring (Miller and Niemeyer, 1995). All the unc-4::gfp-positive cells in the ventral cord also stained with anti-ceo/e, indicating that CeO/E is expressed in all DA neurons. In contrast, the SAB ring interneurons did not stain with anti-ceo/e. GFP reporter expression in unc-3 worms The levels of expression of genes known to function in ASI chemosensory neurons or ventral cord motor neurons was visualized using GFP reporter constructs in wild-type and unc-3 mutants. These GFP reporters, including the unc-3::gfp fusion, were also used to study the morphology of the axonal projections in unc-3 worms in comparison to wild-type worms expressing the same GFP construct. N2 males were crossed to hermaphrodites expressing GFP under the control of promoters of the genes unc-3, unc-4 (Miller and Niemeyer, 1995), unc-5 (J. Culotti, personal communication) and srd-1 (Troemel et al., 1995). Cross-progeny males containing the GFP array were crossed to unc-3(e151) hermaphrodites and Unc-3 F2 progeny were screened for GFP expression. RESULTS Identification of the C. elegans O/E Homologue Using degenerate PCR primers based on sequences conserved between the mammalian O/E proteins and the Drosophila homologue collier (Crozatier et al., 1996), we identified from C. elegans genomic DNA a fragment containing a 15-residue open reading frame with 100% amino acid identity to O/E-1. This fragment was used to isolate genomic and cdna clones encoding a single product, which we named CeO/E for C. elegans O/E homologue (Fig. 1). Fig. 2. Alignment of the CeO/E and mouse O/E-1 protein sequences. Amino acids are indicated by single letter code. Identities are indicated by dots and conserved amino acid substitutions are indicated by vertical lines. Gaps are represented by dashes. No effort was made to align the last 89 amino acids of the CeO/E protein. Underlined amino acids indicate the rhlh domain. Amino acid residues in bold correspond to residues mutated in unc-3 alleles: e151 (TGG TGA), e54 (TAC TAG), p1001 (TGT TAT). The peptide sequence used to generate the anti-ceo/e antibodies are indicated in bold italics. The critical residues (H 160, C 164, 167, 173) of the zinc finger coordination motif (boxed) are indicated by plain italics. CeO/E is as similar to O/E-1 as it is to the other O/E family members. The three members of the O/E family, O/E-1 (Olf-1/EBF), O/E-2 and O/E-3 share 95% overall amino acid identity and possess similar DNA-binding specificity and transactivation ability (Wang et al., 1997). The Drosophila collier protein is no more similar to the mammalian O/E proteins than it is to CeO/E. CeO/E MSLTAPLRAGQMNFYDEPYNPVLNLHIQPSVKDENQRSTWPIIDTSNTSTQIARAHFEKHPP 62 mo/e-1 MFGIQESIQRSGSSMKEEPLGSGMNAVRTWMQGAGVLDANTAAQSGVGLARAHFEKQPP CeO/E NNLRKSNFFHFVIALYDRNSQPIEVERTQFAGFVEKEKEVDGQDTRNGIHYRLSLMFQNGIR 124 mo/e-1 SNLRKSNFFHFVLALYDRQGQPVEIERTAFVGFVEKEKEANSEKTNNGIHYRLQLLYSNGIR p1001 CeO/E SEHDLFVRLIDSSTKQAITYEGQDKNPEMCRVLLTHEVMCSRCCEKKSCGNRNETPSDPVII 186 mo/e-1 TEQDFYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVII CeO/E DRFFLKFFLKCNQNCLKNAGNPRDMRRFQVVLCSSARIDGPLLAVSDNMFVHNNSKHGRRTK 248 mo/e-1 DRFFLKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRAR e151 CeO/E RTDASDDSEYSESAELPSSVPVIKALFPSEGWIQGGTQVVLIGENFFEGLQVAFGTASPNWG 310 mo/e-1 RLDPSE ATPCIKAISPSEGWTTGGATVIIIGDNFFDGLQVIFGT-MLVWS e54 CeO/E ESVQLISPHAIRVTTPPKHSAGPVDVTLQYKSKTYSRGTPLRFSYITLAEPGIEYGFQRLQK 372 mo/e-1 E---LITPHAIRVQTPPRHIPGVVEVTLSYKSKQFCKGTPGRFIYTALNEPTIDYGFQRLQK CeO/E LLPKYPGDPERLPKDQILKRAAELAEALYNRTSTESLSSYYHTQFDATSDYAARTHTSPRST 454 mo/e-1 VIPRHPGDPERLPKEVILKRAADLVEALYGMPHNNQEIILKRAADIAEALYSVPRNHNQLPA CeO/E LPYGAGPPALSSAVYQTSYPTVNATPAANFLNTQTGFATFGAVNPFAATLQSSSRLS* 511 mo/e-1 LANTSVHAGMMGVNSFSGQLAVNVSEASQATNQGFTRNSSSVSPHGYVPSTTPQQTNYNSVT mo/e-1 TSMNGYGSAAMSNLGGSPTFLNGSAANSPYAIVPSSPTMASSTSLPSNCSSSSGIFSFSPAN mo/e-1 MVSAVKQKSAFAPVVRPQTSPPPTCTSTNGNSLQAISGMIVPPM*

4 1564 B. C. Prasad and others The CeO/E and mammalian O/E-1 proteins share ~70% similarity over the first 400 amino acids and greater than 80% similarity in regions previously shown to be essential for homodimerization and DNA binding (Hagman et al., 1993, 1995; Wang and Reed, 1993) (Fig. 2). The CeO/E protein encodes only the first helix of a unique repeat helix-loophelix (rhlh) motif conserved in the mammalian O/E proteins, but also contains upstream sequences that have been suggested as an alternative HLH motif in Drosophila collier (Crozatier et al., 1996) (Fig. 2). The C-terminal domains of the O/E family members are ~50% identical, but share no homology with CeO/E. However, the C terminus of CeO/E retains the serine- (~16%) and proline- (~8%) rich character of the mammalian homologues (Wang and Reed, 1993; Wang et al., 1997). The unc-3 gene encodes CeO/E The CeO/E cdna is partially encoded by the cosmid F42D1 which was mapped in the vicinity of the unc-3 locus on LGX by Sean Eddy (personal communication). The unc-3 locus was defined by mutations that cause worms to move abnormally (uncoordinated phenotype) (Brenner, 1974). Mosaic analysis indicated that the focus of action of the unc-3 locus was in the motor neurons rather than the surrounding hypodermis (Herman, 1987). EM reconstructions showed that motor neurons develop disorganized processes that receive inappropriate synaptic input and form neuromuscular junctions at ectopic sites (Chalfie and White, 1988). To determine whether CeO/E is the product of the unc-3 gene, we attempted to rescue unc-3 mutant worms by transformation with a 12 kb SalI genomic fragment encoding CeO/E (Fig. 1). This construct, when injected into the null allele unc-3(e151), restored wild-type movement to transformed F2 progeny. Heritable rescue of the Unc-3 phenotype was also seen in three independent lines derived from the rescued F2s. To confirm that unc-3 encodes CeO/E, we sequenced the CeO/E coding regions of three unc-3 alleles. We found that unc-3(e151) and unc-3(e54) code for premature stop codons within the regions shown to be essential for dimerization and DNA binding in the mammalian proteins (Hagman et al., 1995; Fig. 2). The third allele, unc-3(p1001), replaced a conserved cysteine with a tyrosine in a zinc finger motif essential for DNA binding (Hagman et al., 1995; Fig. 2). We concluded that unc- 3 encodes the CeO/E protein. Expression of CeO/E in the ventral nerve cord The expression pattern of CeO/E was initially studied by fusing the promoter and the first 55 codons of unc-3 to GFP (Chalfie et al., 1994), and injecting worms with this construct to generate four heritably transformed lines. In all lines, expression of unc-3::gfp was first observed in late stage Fig. 3. Patterns of CeO/E::GFP expression in larvae and adults. (A,B) Wild-type L2 larva with an extrachromosomal unc-3::gfp array. The postembryonic VA and VB motor neurons are clearly seen as clustered pairs of neurons along the ventral cord. Fainter GFP is also detected in other motor neurons. (A) Fluorescence; (B) Nomarski image. (C,D) unc-3(e151) L1 larva with an extrachromosomal unc-3::gfp array. Defects in axonal projections are clearly seen along the ventral cord (arrow). (C) Fluorescence; (D) Nomarski image. (E) Wild-type L4 larva transformed with an extrachromosomal unc-3::gfp array and showing GFP fluorescence in several motor neurons in the ventral cord. The GFP contains a nuclear localization signal. (F) Anterior region of an unc-3(e151) adult hermaphrodite transformed with an extrachromosomal unc-3::gfp array. The ventral nerve cord appears defasiculated with multiple, disorganized axons (arrows). (G,H) Head of an L4 larva transformed with an extrachromosomal unc-3 ::GFP array and showing GFP expression in the ASI chemosensory neurons. (G) The identification of ASI (green) is based on its location with respect to chemosensory neurons visualized by labeling with DiI (red). DiI labels six pairs of amphid neurons: ASK, ADL, AWB, ASH, ASJ, ASI. (H) Double exposure of Nomarski and GFP image of adult head. The ASI cell bodies (arrowhead) are located just anterior to the terminal bulb of the pharynx.

5 unc-3 encodes an O/E transcription factor 1565 embryos (3-fold) in neurons of the ventral cord. In L1 larvae, we counted 16 cells that expressed unc-3::gfp (Table 1) among the 22 embryonic motor neurons present in the ventral cord at this stage (Sulston et al., 1983). By late L1 and early L2, when 57 new motor neurons are added to the ventral cord (Sulston, 1975), we observed an increase in the number of GFP-positive cells (Table 1). In two lines expressing GFP from an extrachromosomal array, we observed up to 41 ventral cord motor neurons expressing the reporter at similar intensity (Fig. 3E; Table 1). In the integrated line examined, the 23 VA and VB motor neurons expressed GFP at high levels, while 3 strongly staining and up to 11 additional faintly expressing cells were also detected (Fig. 3A). High levels of expression were maintained in these neurons through the L3 stage, but diminished in later stages. No GFP expression was observed in the ventral cord of a majority of adult worms. The unc-3::gfp fusion was also used to visualize axons of motor neurons in unc-3(e151) mutant worms. We found that ventral cord motor neuron axons are highly disorganized and often form defasiculated processes not seen in wild-type worms (compare Fig. 3A to Fig. 3C,F). These defects are consistent with those observed in electron microscopic reconstructions of unc-3 mutants (J. White, personal communication). Antibodies directed against the C terminus of CeO/E were used to confirm the GFP reporter experiments and examine the expression of CeO/E in early development. We first detected anti-ceo/e staining in 8 nuclei in the 400- to 550-cell embryo (Fig. 4A), the stage during which the embryonic motor neurons are first formed (Sulston et al., 1983). The CeO/E-expressing cells were located in positions consistent with motor neurons destined for the ventral cord: initially located on the posterior side close to the periphery of the embryo and subsequently converging toward the ventral midline (Fig. 4B). By comma stage, the number of CeO/E-expressing cells increased to 16 and these cells became positioned along the presumptive ventral cord, again consistent with their identification as embryonic motor neurons (Sulston et al., 1983) (Fig. 4C). In late stage embryos and early L1 larvae, 16 cells expressed CeO/E at high levels along the ventral cord, in addition to at least 6 faintly immunoreactive cells also located along the ventral cord in the head and tail regions (Fig. 4D,E). An earlier onset of expression observed by direct visualization of the endogenous protein with specific antibodies than detected using the GFP reporter was consistent with what has been reported in other systems (Heim et al., 1994) and may reflect the absence of critical regulatory elements. To identify the neurons expressing CeO/E, we stained an unc-4::gfp-expressing line with CeO/E antibodies. unc-4::gfp identifies all DA motor neurons in embryos (Miller and Niemeyer, 1995). The presence of CeO/E in all GFPpositive cells located in the ventral cord in this line confirmed that CeO/E is expressed in all DA motor neurons (data not shown). The inhibitory DD embryonic motor neurons, identified based on the position of DAPI-labeled cells along the ventral cord and GFP-positive DA motor neurons, did not appear to express CeO/E at levels that were detectable by antibody staining. These data suggest that the 16 DA and DB embryonic motor neurons express CeO/E at high levels in addition to 6 cells in the head and tail of the worm (Table 1). The number of cells expressing CeO/E increased dramatically in the late L1 stage when postembryonic motor Table 1. Number of nuclei expressing CeO/E in the ventral cord No. of No. of No. of motor neurons in ventral cord* CeO/E::GFP- anti-ceo/e- Stage Embryonic Postembryonic positive nuclei positive nuclei L ±4 L ±5 *Embryonic motor neurons, born 280 minutes into development, include 9 DA, 7 DB and 6 DD. Postembryonic motor neurons are added to the ventral cord in the late L1 stage (11 AS, 12 VA, 11 VB, 13 VD, 6 VC and 4 neurons in the posterior ventral cord (Sulston and Horvitz, 1977).) Averages obtained by counting unc-3:: GFP-expressing cells in 10 or more L1 and L2 animals in one line containing an extrachromosomal array and in one line containing an integrated array. In a second non-integrated line, 37 cells appeared to express unc-3::gfp consistently. Average number of cells staining with the anti-ceo/e antibody in L1 (n=15) and L2 (n=25) worms. neurons descendants of the P neuroblast are added to the ventral cord (Sulston, 1975). The P neuroblast divides to generate hypodermal cells and 3 to 5 neurons, each belonging to a different class of postembryonic ventral cord neurons (VA, VB, VC, VD or AS) (Sulston and Horvitz, 1977). During this time, CeO/E staining in the embryonic motor neurons decreased in intensity. More than 40 ventral cord motor Fig. 4. Immunolocalization of CeO/E. Embryos and larvae were stained with anti-ceo/e polyclonal antibody. In all photos, anterior is to the left, posterior is to the right. Each embryo is approximately 45 µm in length. (A) Ventral view of 400- to 550-cell embryo. 8 positive nuclei are visible. (B) Ventral view of bean-stage embryo with 16 positive nuclei. (C) Lateral view of comma-stage embryo (ventral side down) showing several neurons staining in the region of the ventral cord. An ASI amphid neuron (arrowhead) is also visible in this focal plane. Lateral views of a 3-fold embryo (D) and L1 larva (E) (250 µm in length) showing intense staining in 16 nuclei in the ventral cord. At least 6 additional nuclei along the ventral cord show fainter staining. This pattern is consistent with all 22 embryonic motor neurons expressing CeO/E.

6 1566 B. C. Prasad and others neurons stained prominently in L2 and later stages. We frequently observed clusters of 3 to 5 cells along the ventral cord expressing CeO/E. Because these ventral cord motor neurons generated by the P neuroblast tend to remain clustered, our observation of clusters of up to 5 CeO/E-positive motor neurons is consistent with all 5 classes of postembryonic motor neurons expressing CeO/E. It remains possible, however, that a subset of neurons within each class do not express CeO/E. In summary, expression of CeO/E was observed in most ventral cord motor neurons. The levels of expression were highest during periods of axonal outgrowth: at the embryonic 2-fold stage for embryonic ventral cord motor neurons approximately 480 minutes of development (Durbin, 1987), and at late L1 and early L2 stages for postembryonic ventral cord motor neurons (Sulston, 1975). CeO/E is expressed in ASI chemosensory neurons We observed CeO/E expression in two cells outside of the ventral cord. Using the CeO/E antibody, expression in these cells was first detected in 400 to 550-cell stage in the most anterior region of the embryo. By comma stage, the immunoreactive nuclei were located closer to the ventral cord in a position consistent with that of the ASI amphid neurons (Fig. 4C). These cells continued to stain with anti-ceo/e antibody and expressed unc-3::gfp throughout development (Fig. 3G,H). We confirmed the identity of these cells as the ASI neurons by staining adult worms expressing unc-3::gfp with DiI, a lipophilic dye that stains projections and cell bodies of chemosensory neurons (Fig. 3G,H). The two unc-3::gfpexpressing cells in the head filled with DiI, and their position with respect to the other DiI-positive cells identified them as the ASI amphid neurons. Surprisingly, expression of the unc-3::gfp was either greatly reduced (33/76 worms) or undetectable (35/76 worms) in the ASI amphid neurons of unc-3(e151) mutants although such a reduction in unc-3::gfp expression was not observed in the ventral cord motor neurons (Fig. 3C,F). These observations indicate that CeO/E is expressed in the ASI amphid neurons throughout development and that maintenance of this expression is dependent on unc-3 function. Characterization of ASI amphid neurons in unc-3 mutants The ASI amphid neurons are chemosensory neurons implicated in a number of sensory processes, including dauer formation (Bargmann and Horvitz, 1991; Ren et al., 1996; Schackwitz et al., 1996). The dauer program is an alternative developmental stage that is activated under conditions of crowding, low food and unfavorable temperatures. Previously, unc-3 mutants were noted for their tendency to enter the dauer pathway under inappropriate conditions (M. Ailion and J. Thomas, personal communication). We have confirmed these results: unc-3 mutant worms raised at 27 C exhibit a high frequency of dauer formation in comparison to wild-type worms (Table 2). These observations are consistent with the possibility that unc-3 is required for the proper function and/or development of the ASI neurons. To investigate this possibility further, we examined the expression of an ASI-specific marker in unc-3 mutants. srd-1 is a putative seven transmembrane odorant receptor expressed in ASI (Troemel et al., 1995). We find that >95% of Table 2. DiI filling and Dauer phenotype of unc-3 mutant alleles Strain % dauer at 27 C* % labeled with DiI N >95 unc-3(e151) 98.5 >95 unc-3(e54) unc-3(p1001) 76.5 >95 *Dauer formation was assayed by placing 15 adults on each of two seeded plate for 10 hours at 20 C. The adults were removed from the plates and the number of embryos on the plate were counted to ensure that all plates were equivalent. The plates were placed at 27 C for 72 hours after which the number of dauers were counted. More than 800 progeny worms were assayed for each strain. The numbers represent an average calculated after two independent counts of each plate. DiI filling: worms were dye filled as described in methods. At least 50 worms were counted for each strain. unc-3(e151) mutants expressed an srd-1::gfp fusion. It remains possible that unc-3(e151) is not a genetic null and a stronger unc-3 allele would affect expression of receptors and other genes including srd-1. We also examined whether unc-3 mutants exhibit defects in the ASI dendritic and axonal projections. The trajectory of ASI axonal projections and those of other chemosensory neurons were examined in unc-3(e151) and wild-type worms. We used DiI labeling in three unc-3 alleles and unc-3(e151) worms expressing srd-1::gfp fusion to visualize the ASI projections. In unc-3(e151) and unc- 3(p1001), all chemosensory neurons including the ASI amphid neurons, appeared to dye-fill normally. However, in 50% of unc-3(e54) worms, the ASI amphids were not DiI labeled whereas the remaining 5 pairs of chemosensory neurons dyefilled normally (Table 2). We found that the ASI dendrites bundle as in wild type with the dendrites of other amphid neurons in their trajectory towards the anterior tip (nose) of the worm. Axonal projections into the nerve ring also appeared normal, although we cannot exclude subtle defasiculation defects within the nerve ring. These observations indicate that unc-3 is not generally required for proper outgrowth of ASI projections. Expression of motor neuron markers in unc-3 mutants In an attempt to identify targets regulated by UNC-3 in motor neurons, we tested the expression of unc-4::gfp and unc- 5::GFP reporter fusions in unc-3(e151) worms. UNC-4, a homeodomain transcription factor required for proper synaptic choice by the VA motor neurons (Miller and Niemeyer, 1995), is expressed in the DA, VA and VC motor neurons. UNC-5, an UNC-6/netrin receptor, is involved in axonal pathfinding of the D-type inhibitory motor neurons (J. Culotti, personal communication). Levels of expression of both reporter constructs were not markedly affected in unc-3 mutants suggesting that unc-3 does not control the expression of these two genes in the DA, VA, VC and D class motor neurons. The visualized axons in unc-3 worms, however, displayed modest defasiculation and pathfinding defects consistent with the expression pattern of CeO/E. Reproducible defasiculation defects were observed in DA, VA and VC motor neurons in worms expressing unc-4::gfp (100% of worms showed defects), consistent with unc-3 expression in these cells. In

7 unc-3 encodes an O/E transcription factor 1567 comparison to wild-type worms expressing unc-5::gfp, 41% of the unc-3(e151) worms expressing the same construct showed defects in the inhibitory D class motor neurons. Since CeO/E is not expressed in the embryonic DD motor neurons, these results suggest that unc-3 may lead to secondary axonal projection defects in cells that do not express CeO/E. Direct immunostaining to visualize the axons of D type motor neurons is required to rule out artifacts arising from GFP expression. DISCUSSION Cloning and characterization of unc-3 We have shown that the C. elegans unc-3 gene encodes an homologue of the O/E family of mammalian transcription factors. The CeO/E protein shares high amino acid identity with the mammalian proteins in regions essential for its activity including the dimerization, DNA-binding and transcriptional activation domains (Hagman et al., 1995). The Unc-3 phenotype was heritably rescued when a genomic clone encoding the CeO/E protein was used to transform unc-3(e151) worms. In addition, we mapped the nucleotide changes leading to the Unc-3 phenotype in three mutant alleles to the genomic region encoding the CeO/E protein. The remarkable sequence conservation between the mammalian and C. elegans proteins is accompanied by expression in neurons (sensory and motor neurons, respectively) in a similar temporal pattern. The expression of O/E proteins was also observed throughout development in a pair of ASI chemosensory neurons of worms and olfactory neurons in mammals. Role of unc-3 in the axonogenesis of ventral nerve cord motor neurons High levels CeO/E expression, detected with antibodies, occurs as embryonic and postembryonic ventral cord motor neurons begin to project axons to target tissues and expression is reduced markedly when this process is complete. Electron microscopic reconstructions reveal severe defasiculation defects in the ventral cord of unc-3 mutant worms suggesting a role in axonal pathfinding for CeO/E. The expression in the ventral cord motor neurons parallels the expression of O/E family members in the developing mouse spinal cord, where these proteins also display a similar temporal expression pattern. The O/E proteins are first detected at E11.5 when spinal cord neurons begin to project to target tissues and remain elevated until postnatal day 1 when axonogenesis is largely complete (Davis and Reed, 1996). Downstream targets in spinal cord neurons that are regulated by the mammalian O/E proteins have not been identified. Several mutations in C. elegans have been described that specifically affect the axonal outgrowth phase of neuronal differentiation. The unc-30 (Jin et al., 1994) and unc-4 (Miller and Niemeyer, 1995) genes encode homeodomain transcription factors that apparently regulate the expression of unidentified proteins essential for axonal pathfinding. The Drosophila lola and logo (Giniger et al., 1994) genes encode transcription factors that play a role in specific axon patterning in the developing nervous system. The CeO/E protein, a putative transcription factor required for axonal pathfinding, with highly conserved homologues in vertebrates (Hagman et al., 1993; Wang and Reed, 1993; Wang et al., 1997) and invertebrates (Crozatier et al., 1996) has been implicated in having a pioneering role in axonal pathfinding. The requirement for unc-3 in this process (Wightman et al., 1997) suggests that CeO/E might directly control the expression of genes that are involved in growth cone pioneering along the ventral cord, fasiculation or directing commisural projections. Alternatively, unc-3 might indirectly influence the expression of these genes through a transcriptional regulatory cascade. The isolation of CeO/E-regulated targets can potentially be extended to the identification of related proteins that play critical roles in mammalian axonal pathfinding. CeO/E activity in ASI chemosensory neurons In contrast to the transient expression of CeO/E in the ventral cord, CeO/E is expressed throughout development in the ASI chemosensory neurons. A temperature-sensitive Daf-c phenotype has been associated with the unc-3 mutations. Laser ablation studies (Bargmann et al., 1990; Bargmann and Horvitz, 1991), in addition to the expression of a key dauer pathway signaling molecule, daf-7, expressed exclusively in ASI, suggest that neuronal activity in the ASI amphid neurons suppresses dauer formation. Although the Daf-c phenotype observed in unc-3 mutants could result from the miswiring of ASI axonal projections and failure to deliver a tonic inhibitory signal to the appropriate target cell, our data suggest that the dauer phenotype does not result from simple miswiring. DiI filling of unc-3 mutants indicated that the axonal and dendritic projections of the ASI amphid neurons in at least two alleles were normal. Instead, CeO/E might regulate expression of downstream components essential for transducing environmental cues and stimulating entrance into the dauer pathway. CeO/E may have distinct functions in axonal pathfinding and regulation of signal transduction components. The Daf-c phenotype of unc-3 alleles is consistent with predicted severity of the mutations found in each allele. Interestingly, unc-3(e54), which makes a protein 60 amino acids longer than unc-3(e151) and has a somewhat less penetrant Daf-c phenotype, also has dye-filling defects in 50% of the worms, suggesting a possible axonal and/or dendritic defect in these worms. This could be explained by hypothesizing that the unc-3(e54) protein, lacking a transcriptional activation domain, is still able to dimerize and bind DNA and thus causes transcriptional repression rather than activation of specific genes involved in axonal pathfinding. As a result, unc-3(e54) might have a phenotype in ASI axonal pathfinding stronger than an unc-3 deficiency. Presumably, at other sites, like those required to elicit the Dafc phenotype, this defective dimer would not be able to elicit the inhibitory effect. Alternatively, there may exist differences in the strains that contribute to the phenotype observed. The continual expression of CeO/E in the ASI amphid neurons long after synaptic connections are complete is similar to the pattern seen in mammalian olfactory neurons where expression in these cells remains elevated throughout development. The presence of O/E-binding sites proximal to olfactory-specific genes suggests that O/E proteins regulate the odorant signal transduction cascade. The CeO/E protein might likewise regulate expression of genes involved in control of entry into the dauer pathway and other signaling cascades in the ASI amphid neurons.

8 1568 B. C. Prasad and others In conclusion, we have demonstrated that the C. elegans unc-3 gene encodes the CeO/E protein. This protein is expressed in the motor neurons of the ventral nerve cord and ASI chemosensory neurons. The expression pattern of the protein, in correlation with genetic data available, suggests that CeO/E might be involved in axonal pathfinding in the ventral cord and control expression of genes that stimulate entry into the dauer pathway. The mammalian O/E family of transcription factors were identified based on their binding to cis-acting regulatory elements in a collection of coordinately expressed neuronal genes. The identification of the unc-3 gene product as CeO/E may provide a direct approach to identify relevant downstream targets. We thank Harald Hutter, Andy Fire, Cori Bargmann, Adam Antebi, Greg Cost, and members of the Reed and Seydoux laboratories. We also thank John White, Joe Culotti, Takao Inoue, Mike Aillion, Jim Thomas and Sean Eddy for sharing prepublication results and providing necessary strains and constructs. REFERENCES Bargmann, C. I. and Horvitz, H. R. (1991). Control of larval development by chemosensory neurons in Caenorhabditis elegans. Science 251, Bargmann, C. I., Thomas, J. H. and Horvitz, H. R. (1990). Chemosensory cell function in the behavior and development of Caenorhabditis elegans. Cold Spring Harbor Symposia on Quantitative Biology Volume LV, Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, Chalfie, M. and White, J. (1988). The nervous system. In The Nematode Caenorhabditis elegans Plainview, NY: Cold Spring Harbor Laboratory. Crozatier, M., Valle, D., Dubois, L., Ibnsouda, S. and Vincent, A. (1996). collier, a novel regulator of Drosophila head development, is expressed in a single mitotic domain. Current Biol. 6, Davis, J. A. and Reed, R. R. (1996). Role of Olf-1 and Pax-6 transcription factors in neurodevelopment. J. Neurosci. 16, Durbin, R. M. (1987). Studies on the development and organization of the nervous system of C. elegans. PhD Thesis, University of Cambridge, UK. Giniger, E., Tietje, K., Jan, L. Y. and Jan, Y. N. (1994). lola encodes a putative transcription factor required for axon growth and guidance in Drosophila. Development 120, Hagman, J., Belanger, C., Travis, A., Turck, C. W. and Grosschedl, R. (1993). Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression. Genes Dev. 7, Hagman, J., Gutch, M. J., Lin, H. and Grosschedl, R. (1995). EBF contains a novel zinc coordination motif and multiple dimerization and transcriptional activation domains. EMBO J. 14, Heim, R., Prasher, D. and Tsien, R. (1994). Wavelength mutations and posttranslational auto-oxidation of green fluorescent protein. Proc. Natl Acad. Sci. USA 91, Herman, R. K. (1987). Mosaic analysis of two genes that affect nervous system structure in Caenorhabditis elegans. Genetics 116, Hodgkin, J. (1997). Genetics. C. elegans II. pp Plainview: Cold Spring Harbor Laboratory Press. Jin, Y., Hoskins, R. and Horwitz, H. R. (1994). Control of type-d GABAergic neuron differentiation by C. elegans UNC-30 homeodomain protein. Nature 372, Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C. elegans: Extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, Miller, D. M. and Niemeyer, C. J. (1995). Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input. Development 121, Miller, D. M. and Shakes, D. C. (1995). Immunofluoresence microscopy. In Caenorhabditis elegans: Modern Biological Analysis of an Organism San Diego: Academic Press. Ren, P., Lim, C.-S., Johnsen, R., Albert, P. S., Pilgrim, D. and Riddle, D. L. (1996). Control of C. elegans larval development by neuronal expression of a TGF-β homologue. Science 274, Ruvkun, G. (1997). Patterning the nervous system. In C. elegans II. Plainview: Cold Spring Harbor Laboratory Press. Schackwitz, W. S., Inoue, T. and Thomas, J. H. (1996). Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans. Neuron 17, Sulston, J. E. (1975). Post-embryonic development in the ventral cord of Caenorhabditis elegans. Philosoph. Trans. Roy. Soc. Lond. (B) 275, Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Dev. Biol. 56, Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, Troemel, E. R., Chou, J. H., Dwyer, N. D., Colbert, H. A. and Bargmann, C. I. (1995). Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, Wang, M. M. and Reed, R. R. (1993). Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364, Wang, S., Tsai, R. Y. L. and Reed, R. R. (1997). The Characterization of the Olf-1/EBF-like HLH transcription factor family: implications in olfactory gene regulation and neuronal development. J. Neurosci. 17, White, J. G., Southgate, E., Thomson, J. N. and Brenner, S. (1975). The Structure of the ventral nerve cord of Caenorhabditis elegans. Philosoph. Trans. Roy. Soc. Lond. (B) 275, White, J. G., Southgate, E., Thomson, J. N. and Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philosoph. Trans. Roy. Soc. Lond. (B) 314, Wightman, B., Baran, R. and Garriga, G. (1997). Genes that guide growth cones along the C. elegans ventral nerve cord. Development124,