The directed migration of gonadal distal tip cells in Caenorhabditis elegans requires NGAT-1, a ß1,4-N-acetylgalactosaminyltransferase enzyme.

Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted ß1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC). An amino-terminal partial deletion of ngat-1 causes a similar but lower penetrance ts phenotype. The existence of multiple ts alleles with distinctly different molecular DNA lesions, neither of which is likely to encode a ts protein, indicates that NGAT-1 normally prevents innate temperature sensitivity for phase 2 DTC pathfinding. Temperature shift analyses indicate that the ts period for the ngat-1 mutant defect ends by the beginning of post-embryonic development-nearly 3 full larval stages prior to the defective phase 2 migration affected by ngat-1 mutations. NGAT-1 homologs generate glycan-terminal GalNAc-ß1-4GlcNAc, referred to as LacdiNAc modifications, on glycoproteins and glycolipids. We also found that the absence of the GnT1/Mgat1 activity [UDP-N-acetyl-D-glucosamine:α-3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase 1 (encoded by C. elegans gly-12, gly-13, and gly-14 and homologous to vertebrate GnT1/Mgat1)], causes a similar spectrum of DTC phenotypes as ngat-1 mutations-primarily affecting posterior DTC phase 2 migration and preventing manifestation of the same innate ts period as ngat-1. GnT1/Mgat1 is a medial Golgi enzyme known to modify mannose residues and initiate N-glycan branching, an essential step in the biosynthesis of hybrid, paucimannose and complex-type N-glycans. Quadruple mutant animals bearing putative null mutations in ngat-1 and the three GnT genes (gly-12, gly-13, gly-14) were not enhanced for DTC migration defects, suggesting NGAT-1 and GnT1 act in the same pathway. These findings suggest that GnTI generates an N-glycan substrate for NGAT-1 modification, which is required at restrictive temperature (25°C) to prevent, stabilize, reverse or compensate a perinatal thermo-labile process (or structure) causing late larval stage DTC phase 2 migration errors.

Forty-five years of cell-cycle genetics.

In the early 1970s, studies in Leland Hartwell's laboratory at the University of Washington launched the genetic analysis of the eukaryotic cell cycle and set the path that has led to our modern understanding of this centrally important process. This 45th-anniversary Retrospective reviews the steps by which the project took shape, the atmosphere in which this happened, and the possible morals for modern times. It also provides an up-to-date look at the 35 original CDC genes and their human homologues.

The Wnt Frizzled Receptor MOM-5 Regulates the UNC-5 Netrin Receptor through Small GTPase-Dependent Signaling to Determine the Polarity of Migrating Cells.

Wnt and Netrin signaling regulate diverse essential functions. Using a genetic approach combined with temporal gene expression analysis, we found a regulatory link between the Wnt receptor MOM-5/Frizzled and the UNC-6/Netrin receptor UNC-5. These two receptors play key roles in guiding cell and axon migrations, including the migration of the C. elegans Distal Tip Cells (DTCs). DTCs migrate post-embryonically in three sequential phases: in the first phase along the Antero-Posterior (A/P) axis, in the second, along the Dorso-Ventral (D/V) axis, and in the third, along the A/P axis. Loss of MOM-5/Frizzled function causes third phase A/P polarity reversals of the migrating DTCs. We show that an over-expression of UNC-5 causes similar DTC A/P polarity reversals and that unc-5 deficits markedly suppress the A/P polarity reversals caused by mutations in mom-5/frizzled. This implicates MOM-5/Frizzled as a negative regulator of unc-5. We provide further evidence that small GTPases mediate MOM-5's regulation of unc-5 such that one outcome of impaired function of small GTPases like CED-10/Rac and MIG-2/RhoG is an increase in unc-5 function. The work presented here demonstrates the existence of cross talk between components of the Netrin and Wnt signaling pathways and provides further insights into the way guidance signaling mechanisms are integrated to orchestrate directed cell migration.

Netrins and Wnts function redundantly to regulate antero-posterior and dorso-ventral guidance in C. elegans.

Guided migrations of cells and developing axons along the dorso-ventral (D/V) and antero-posterior (A/P) body axes govern tissue patterning and neuronal connections. In C. elegans, as in vertebrates, D/V and A/P graded distributions of UNC-6/Netrin and Wnts, respectively, provide instructive polarity information to guide cells and axons migrating along these axes. By means of a comprehensive genetic analysis, we found that simultaneous loss of Wnt and Netrin signaling components reveals previously unknown and unexpected redundant roles for Wnt and Netrin signaling pathways in both D/V and A/P guidance of migrating cells and axons in C. elegans, as well as in processes essential for organ function and viability. Thus, in addition to providing polarity information for migration along the axis of their gradation, Wnts and Netrin are each able to guide migrations orthogonal to the axis of their gradation. Netrin signaling not only functions redundantly with some Wnts, but also counterbalances the effects of others to guide A/P migrations, while the involvement of Wnt signaling in D/V guidance identifies Wnt signaling as one of the long sought mechanisms that functions in parallel to Netrin signaling to promote D/V guidance of cells and axons. These findings provide new avenues for deciphering how A/P and D/V guidance signals are integrated within the cell to establish polarity in multiple biological processes, and implicate broader roles for Netrin and Wnt signaling--roles that are currently masked due to prevalent redundancy.

Genetics of extracellular matrix remodeling during organ growth using the Caenorhabditis elegans pharynx model.

The organs of animal embryos are typically covered with an extracellular matrix (ECM) that must be carefully remodeled as these organs enlarge during post-embryonic growth; otherwise, their shape and functions may be compromised. We previously described the twisting of the Caenorhabditis elegans pharynx (here called the Twp phenotype) as a quantitative mutant phenotype that worsens as that organ enlarges during growth. Mutations previously known to cause pharyngeal twist affect membrane proteins with large extracellular domains (DIG-1 and SAX-7), as well as a C. elegans septin (UNC-61). Here we show that two novel alleles of the C. elegans papilin gene, mig-6(et4) and mig-6(sa580), can also cause the Twp phenotype. We also show that overexpression of the ADAMTS protease gene mig-17 can suppress the pharyngeal twist in mig-6 mutants and identify several alleles of other ECM-related genes that can cause or influence the Twp phenotype, including alleles of fibulin (fbl-1), perlecan (unc-52), collagens (cle-1, dpy-7), laminins (lam-1, lam-3), one ADAM protease (sup-17), and one ADAMTS protease (adt-1). The Twp phenotype in C. elegans is easily monitored using light microscopy, is quantitative via measurements of the torsion angle, and reveals that ECM components, metalloproteinases, and ECM attachment molecules are important for this organ to retain its correct shape during post-embryonic growth. The Twp phenotype is therefore a promising experimental system to study ECM remodeling and diseases.

Genetic association of the GDNF alpha-receptor genes with schizophrenia and clozapine response.

GDNF (glial-cell-line derived neurotrophic factor) is a potent neurotrophic factor for dopaminergic neurons. Neuropsychiatric diseases and their treatments are associated with alterations in the levels of both GDNF and its receptor family (GDNF family receptor alpha or GFRA). GFRA1, GFRA2 and GFRA3 are located in chromosomal regions with suggestive linkage to schizophrenia. In this study we analyzed polymorphisms located in all four known GFRA genes and examined association with schizophrenia and clozapine response. We examined SNPs across the genes GFRA1-4 in 219 matched case-control subjects, 85 small nuclear families and 140 schizophrenia patients taking clozapine for 6months. We observed that GFRA3 rs11242417 and GFRA1 rs11197557 variants were significantly associated with schizophrenia after combining results from both schizophrenia samples. Furthermore, we found an overtransmission of the G-C GFRA1 rs7920934-rs730357 haplotype to subjects with schizophrenia and association of A-T-G-G GFRA3 rs10036665-rs10952-rs11242417-rs7726580 with schizophrenia in the case-control sample. On the other hand, GFRA2 variants were not associated with schizophrenia diagnosis but subjects carrying T-G-G rs1128397-rs13250096-rs4567028 haplotype were more likely to respond to clozapine treatment. The statistical significance of results survived permutation testing but not Bonferroni correction. We also found nominally-significant evidence for interactions between GFRA1, 2 and 3 associated with schizophrenia and clozapine response, consistent with the locations of these three genes within linkage regions for schizophrenia.

Dopamine counteracts octopamine signalling in a neural circuit mediating food response in C. elegans.

Animals assess food availability in their environment by sensory perception and respond to the absence of food by changing hormone and neurotransmitter signals. However, it is largely unknown how the absence of food is perceived at the level of functional neurocircuitry. In Caenorhabditis elegans, octopamine is released from the RIC neurons in the absence of food and activates the cyclic AMP response element binding protein in the cholinergic SIA neurons. In contrast, dopamine is released from dopaminergic neurons only in the presence of food. Here, we show that dopamine suppresses octopamine signalling through two D2-like dopamine receptors and the G protein Gi/o. The D2-like receptors work in both the octopaminergic neurons and the octopamine-responding SIA neurons, suggesting that dopamine suppresses octopamine release as well as octopamine-mediated downstream signalling. Our results show that C. elegans detects the absence of food by using a small neural circuit composed of three neuron types in which octopaminergic signalling is activated by the cessation of dopamine signalling.

C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV.

The gonad arms of C. elegans hermaphrodites acquire invariant shapes by guided migrations of distal tip cells (DTCs), which occur in three phases that differ in the direction and basement membrane substrata used for movement. We found that mig-6 encodes long (MIG-6L) and short (MIG-6S) isoforms of the extracellular matrix protein papilin, each required for distinct aspects of DTC migration. Both MIG-6 isoforms have a predicted N-terminal papilin cassette, lagrin repeats and C-terminal Kunitz-type serine proteinase inhibitory domains. We show that mutations affecting MIG-6L specifically and cell-autonomously decrease the rate of post-embryonic DTC migration, mimicking a post-embryonic collagen IV deficit. We also show that MIG-6S has two separable functions - one in embryogenesis and one in the second phase of DTC migration. Genetic data suggest that MIG-6S functions in the same pathway as the MIG-17/ADAMTS metalloproteinase for guiding phase 2 DTC migrations, and MIG-17 is abnormally localized in mig-6 class-s mutants. Genetic data also suggest that MIG-6S and non-fibrillar network collagen IV play antagonistic roles to ensure normal phase 2 DTC guidance.

UNC-129 regulates the balance between UNC-40 dependent and independent UNC-5 signaling pathways.

The UNC-5 receptor mediates axon repulsion from UNC-6/netrin through UNC-40 dependent (UNC-5 + UNC-40) and independent (UNC-5 alone) signaling pathways. It has been shown that UNC-40-dependent signaling is required for long-range repulsion of UNC-6/netrin; however, the mechanisms used to regulate distinct UNC-5 signaling pathways are poorly understood. We found that the C. elegans transforming growth factor beta (TGF-beta) family ligand UNC-129, graded opposite to UNC-6/netrin, functions independent of the canonical TGF-beta receptors to regulate UNC-5 cellular responses. Our observations indicates that UNC-129 facilitates long-range repulsive guidance of UNC-6 by enhancing UNC-5 + UNC-40 signaling at the expense of UNC-5 alone signaling through interaction with the UNC-5 receptor. This increases the set point sensitivity of growth cones to UNC-6/netrin as they simultaneously migrated up the UNC-129 gradient and down the UNC-6 gradient. Similar regulatory interactions between oppositely graded extracellular cues may be a common theme in guided cell and axon migrations.

Dopamine suppresses octopamine signaling in C. elegans: possible involvement of dopamine in the regulation of lifespan.

Amine neurotransmitters, such as dopamine, serotonin, and noradrenaline, play important roles in the modulation of behaviors and metabolism of animals. InC. elegans, it has been shown that serotonin and octopamine, an invertebrate equivalent of noradrenaline, also regulate lifespan through a mechanism related to food deprivation-mediated lifespan extension. We have shown recently that dopamine signaling, activated by the tactile perception of food, suppresses octopamine signaling and that the cessation of dopamine signaling in the absence of food leads to activation of octopamine signaling. Here, we discuss the apparent conservation of neural and molecular mechanisms for dopamine regulation of octopamine/noradrenaline signaling and a possible role for dopamine in lifespan regulation.

Examination of neurons in wild type and mutants of Caenorhabditis elegans using antibodies to horseradish peroxidase.

Antibodies to horseradish peroxidase (HRP) recognize 27 of 302 neurons and several non-neuronal cells in adult hermaphrodites of the soil nematode Caenorhabditis elegans and can be used to label these cells for cytological analysis in whole animals. The antibodies bind to the anterior members, but not to the posterior members of a set of mechanosensory neurons in wild type animals. Binding to one of the posterior mechanosensory neurons (PVM) occurs when this neuron migrates to an abnormal anterior position in mab-5 mutant animals, suggesting that expression of the epitope recognized by these antibodies is position dependent or that mab-5 mutations transform PVM into AVM intrinsically. The antibodies were used to characterize morphologies of two pairs of lumbar neurons (PHC and PVN) in uncoordinated mutants representing 95 unc genes. PHC and PVN morphologies were normal in most of the unc mutants examined, however, in mutants of 9 unc genes (unc-6, unc-13, unc-33, unc-44, unc-51, unc-61, unc-71, unc-73, and unc-98), misdirected PHC and/or PVN processes were observed at a high frequency. The morphologies of 2 other lumbar neurons, PHA and PHB, were determined previously in these mutants (Hedgecock et al., 1985). Mutations in most, but not all of these 9 unc genes affect the growth of the embryonic lumbar neurons PHA and PHB differently than they affect the growth of the postembryonic lumbar neurons PHC and PVN, indicating that these neurons require different, but overlapping sets of genes for different stages of normal growth and guidance.

The slit receptor EVA-1 coactivates a SAX-3/Robo mediated guidance signal in C. elegans.

The SAX-3/roundabout (Robo) receptor has SLT-1/Slit-dependent and -independent functions in guiding cell and axon migrations. We identified enhancer of ventral-axon guidance defects of unc-40 mutants (EVA-1) as a Caenorhabditis elegans transmembrane receptor for SLT-1. EVA-1 has two predicted galactose-binding ectodomains, acts cell-autonomously for SLT-1/Slit-dependent axon migration functions of SAX-3/Robo, binds to SLT-1 and SAX-3, colocalizes with SAX-3 on cells, and provides cell specificity to the activation of SAX-3 signaling by SLT-1. Double mutants of eva-1 or slt-1 with sax-3 mutations suggest that SAX-3 can (when slt-1 or eva-1 function is reduced) inhibit a parallel-acting guidance mechanism, which involves UNC-40/deleted in colorectal cancer.

C. elegans seu-1 encodes novel nuclear proteins that regulate responses to UNC-6/netrin guidance cues.

In C. elegans, ectopic expression of the UNC-5 netrin receptor is sufficient to cause repulsion of growth cones and cells away from ventral sources of the UNC-6/netrin guidance cue. A genetic suppressor screen identified the seu-1 gene as required for repulsion of touch neuron growth cones ectopically expressing unc-5. We report here that seu-1 mutations also enhance the frequency of distal tip cell migrations of unc-5 or unc-40 mutants. The seu-1 gene encodes two novel proteins (SEU-1A and SEU-1B) containing a charged central domain and several regions of low amino acid complexity. Transgenic rescue experiments indicate that seu-1 can act cell autonomously in the touch neurons and distal tip cells and that SEU-1 function requires both the SEU-1A and SEU-1B isoforms. A GFP fusion construct was expressed in a dynamic pattern throughout development and localized in the nuclei of neuronal and non-neuronal cells, including gonadal leader cells. These results implicate nuclear SEU-1 in the interpretation of UNC-6/netrin directional information by migrating growth cones and cells.

VAB-8, UNC-73 and MIG-2 regulate axon polarity and cell migration functions of UNC-40 in C. elegans.

One of the most intriguing features of axons is their ability to pioneer precise paths to their targets. How guidance-cue information is interpreted and integrated to form intricate neuronal networks has not been fully deciphered. Using Caenorhabditis elegans, we show that highly conserved receptors that guide pioneer axons along the dorsoventral axis, such as UNC-40 and SAX-3 (receptors for UNC-6 and SLT-1 guidance cues, respectively), can be co-opted to affect axon and cell migrations along the anterior-posterior axis. We further identify the kinesin-related VAB-8 protein as an upstream regulator of UNC-40, illuminating VAB-8's mechanism of action in determining the polarity of cell and axon migration. Finally, we show that UNC-73 and its target MIG-2 function with VAB-8 as upstream regulators of UNC-40 and that MIG-2 activity specifies UNC-40 subcellular localization. These data are indicative of previously unidentified regulatory roles for VAB-8 and small GTPases, which act together to regulate guidance receptor functions.

Vulva morphogenesis involves attraction of plexin 1-expressing primordial vulva cells to semaphorin 1a sequentially expressed at the vulva midline.

Vulva development in C. elegans involves cell fate specification followed by a morphogenesis phase in which homologous mirror image pairs within a linear array of primordial vulva cells form a crescent shape as they move sequentially towards a midline position within the array. The homologous pairs from opposite half vulvae in fixed sequence fuse with one another at their leading tips to form ring-shaped (toroidal) cells stacked in precise alignment one atop the other. Here, we show that the semaphorin 1a SMP-1, and its plexin receptor PLX-1, are required for the movement of homologous pairs of vulva cells towards this midline position. SMP-1 is upregulated on the lumen membrane of each primordial vulva cell as it enters the forming vulva and apparently attracts the next flanking homologous PLX-1-expressing vulva cells towards the lumen surface of the ring. Consequently, a new ring-shaped cell forms immediately ventral to the previously formed ring. This smp-1- and plx-1-dependent process repeats until seven rings are stacked along the dorsoventral axis, creating a common vulva lumen. Ectopic expression of SMP-1 suggests it has an instructive role in vulva cell migration. At least two parallel acting pathways are required for vulva formation: one requires SMP-1, PLX-1 and CED-10; and another requires the MIG-2 Rac GTPase and its putative activator UNC-73.

Conversion of cell movement responses to Semaphorin-1 and Plexin-1 from attraction to repulsion by lowered levels of specific RAC GTPases in C. elegans.

Plexins are functional receptors for Semaphorin axon guidance cues. Previous studies have established that some Plexins directly bind RAC(GTP) and RHO. Recent work in C. elegans showed that semaphorin 1 (smp-1 and smp-2) and plexin 1 (plx-1) are required to prevent anterior displacement of the ray 1 cells in the male tail (Fujii et al., 2002; Ginzburg et al., 2002). We show genetically that plx-1 is part of the same functional pathway as smp-1 and smp-2 for male ray positioning. RAC GTPase genes mig-2 and ced-10 probably function redundantly, whereas unc-73, which encodes a GEF for both of these GTPases, is required cell autonomously for preventing anterior displacement of ray 1 cells. RNAi analysis indicates that rho-1-encoded RHO GTPase, plus let-502 and K08B12.5-encoded RHO-kinases, are also required to prevent anterior displacement of ray 1 cells, suggesting that different kinds of RHO-family GTPases act similarly in ray 1 positioning. At low doses of wild-type mig-2 and ced-10, the Semaphorin 1 proteins no longer act through PLX-1 to prevent anterior displacements of ray 1, but have the opposite effect, acting through PLX-1 to mediate anterior displacements of ray 1. These results suggest that Plexin 1 senses levels of distinct RHO and RAC GTPases. At normal levels of RHO and RAC, Semaphorin 1 proteins and PLX-1 prevent a forward displacement of ray 1 cells, whereas at low levels of cycling RAC, Semaphorin 1 proteins and PLX-1 actively mediate their anterior displacement. Endogenously and ectopically expressed SMP-1 and SMP-2 suggest that the hook, a major source of Semaphorin 1 proteins in the male tail, normally attracts PLX-1-expressing ray 1 cells.

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans.

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.

UNC-52/perlecan affects gonadal leader cell migrations in C. elegans hermaphrodites through alterations in growth factor signaling.

The unc-52 gene of Claenorhabditis elegans encodes a homologue of the basement membrane heparan sulfate proteoglycan perlecan. Viable alleles reduce the abundance of UNC-52 in late larval stages and increase the frequency of distal tip cell (DTC) migration defects caused by mutations disrupting the UNC-6/netrin guidance system. These unc-52 alleles do not cause circumferential DTC migration defects in an otherwise wild-type genetic background. The effects of unc-52 mutations on DTC migrations are distinct from effects on myofilament organization and can be partially suppressed by mutations in several genes encoding growth factor-like molecules, including EGL-17/FGF, UNC-129/TGF-beta, DBL-1/TGF-beta, and EGL-20/WNT. We propose that UNC-52 serves dual roles in C. elegans larval development in the maintenance of muscle structure and the regulation of growth factor-like signaling pathways.

Semaphorin 1a and semaphorin 1b are required for correct epidermal cell positioning and adhesion during morphogenesis in C. elegans.

The semaphorin family comprises secreted and transmembrane proteins involved in axon guidance and cell migration. We have isolated and characterized deletion mutants of C. elegans semaphorin 1a (Ce-sema-1a or smp-1) and semaphorin 1b (Ce-sema-1b or smp-2) genes. Both mutants exhibit defects in epidermal functions. For example, the R1.a-derived ray precursor cells frequently fail to change anterior/posterior positions completely relative to their sister tail lateral epidermal precursor cell R1.p, causing ray 1 to be formed anterior to its normal position next to ray 2. The ray cells, which normally separate from the lateral tail seam cell (SET) at the end of L4 stage, remains connected to the SET cell even in adult mutant males. The ray 1 defects are partially penetrant in each single Ce-sema-1 mutant at 20 degrees C, but are greatly enhanced in Ce-sema-1 double mutants, suggesting that Ce-Sema-1a and Ce-Sema-1b function in parallel to regulate ray 1 position. Both mutants also have defects in other aspects of epidermal functions, including head and tail epidermal morphogenesis and touch cell axon migration, whereas, smp-1 mutants alone have defects in defecation and brood size. A feature of smp-1 mutants that is shared with mutants of mab-20 (which encodes Sema-2a) is the abnormal perdurance of contacts between epidermal cells.

The Caenorhabditis elegans gene, gly-2, can rescue the N-acetylglucosaminyltransferase V mutation of Lec4 cells.

UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-TV) is a regulator of polylactosamine-containing N-glycans and is causally involved in T cell regulation and tumor metastasis. The Caenorhabditis elegans genome contains a single orthologous gene, gly-2, that is transcribed and encodes a 669-residue type II membrane protein that is 36.7% identical to mammalian GlcNAc-TV (Mgat-5). Recombinant GLY-2 possessed GlcNAc-TV activity when assayed in vitro, and protein truncations demonstrated that the N-terminal boundary of the catalytic domain is Ile-138. gly-2 complemented the Phaseolus vulgaris leucoagglutinin binding defect of Chinese hamster ovary Lec4 cells, whereas GLY-2(L116R), an equivalent mutation to that which causes the Lec4A phenotype, could not. We conclude that the worm gene is functionally interchangeable with the mammalian form. GlcNAc-TV activity was detected in wild-type animals but not those homozygous for a deletion allele of gly-2. Activity was restored in mutant animals by an extrachromosomal array that encompassed the gly-2 gene. Green fluorescent protein reporter transgenes driven by the gly-2 promoter were expressed by developing embryos from the late comma stage onward, present in a complex subset of neurons in larvae and, in addition, the spermathecal and pharyngeal-intestinal valves and certain vulval cells of adults. However, no overt phenotypes were observed in animals homozygous for deletion alleles of gly-2.