Ji Ying Sze, Yanxia Liu and Gary Ruvkun* SUMMARY

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1 Development 124, (1997) Printed in Great Britain The Company of Biologists Limited 1997 DEV VP16-activation of the C. elegans neural specification transcription factor UNC-86 suppresses mutations in downstream genes and causes defects in neural migration and axon outgrowth Ji Ying Sze, Yanxia Liu and Gary Ruvkun* Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston MA 02114, USA *Author for correspondence ( SUMMARY The POU homeobox gene unc-86 specifies many neuroblast and neural fates in the developing C. elegans nervous system. Genes regulated by unc-86 are mostly unknown. Here we describe a genetic strategy for the identification of downstream pathways regulated by unc-86. We activate UNC-86 transcription activity by inserting the VP16 activation domain into an unc-86 genomic clone that bears all regulatory sequences necessary for normal expression in C. elegans. unc-86/vp16 complements unc-86 mutations in the specification of neuroblast and neural cell fates, but displays novel genetic activities: it can suppress non-null mutations in the downstream genes mec-3 and mec-7 that are necessary for mechanosensory neuron differentiation and function. These data suggest that UNC-86/VP16 increases the expression of mec-3 and mec-7 to compensate for the decreased activities of mutant MEC-3 or MEC-7 proteins. The suppression of mutations in downstream genes by an activated upstream transcription factor should be a general strategy for the identification of genes in transcriptional cascades. unc-86/vp16 also causes neural migration and pathfinding defects and novel behavioral defects. Thus, increased or unregulated expression of genes downstream of unc-86 can confer novel neural phenotypes suggestive of roles for unc-86-regulated genes in neural pathfinding and function. Genetic suppression of these unc- 86/VP16 phenotypes may identify the unc-86 downstream genes that mediate these events in neurogenesis. Key words: neural development, VP16 activation, POU gene, downstream genes, genetic suppression INTRODUCTION The development of the nervous system involves the generation of distinct neuron types with precise synaptic connectivities and signaling pathways. A variety of transcription factors has been shown to control the generation of neurons (Finney et al., 1988; Miller et al., 1992; Salser et al., 1993; Jarman et al., 1993; Erkman et al., 1996), as well as the detailed features of neural type, such as pathfinding (Lundgren et al., 1995), patterns of synaptic connectivity (Miller et al., 1992), neurotransmitter production (Johnson and Hirsh, 1990; McIntire et al., 1993; Jin et al., 1994), and the expression of signaling pathways in particular neural types (Sengupta et al., 1994). These neuronal transcription factors are thought to regulate the expression of a cascade of downstream genes that actually mediate the complex recognition and signaling events during neurogenesis and neural function, such as the expression of surface receptors, signaling molecules and synaptic components (Lewin, 1994). The C. elegans unc-86 gene acts in such a neurogenic regulatory pathway. unc-86 loss-of function mutations perturb neuronal cell lineages by causing neuroblast daughter cells to reiterate the patterns of mother neuroblast cell lineage (Chalfie et al., 1981). These cell lineage defects cause missing mechanosensory neurons and the production of extra dopaminergic sensory neurons (Chalfie et al., 1981). In addition, unc- 86 mutations cause defects in the differentiation of particular neurons. For example, the hermaphrodite-specific neuron (HSN) fails to accumulate the neurotransmitter serotonin (Desai et al., 1988). These neural defects cause behavioral abnormalities, most notably in mechanosensation, egg-laying, chemotaxis, and thermotaxis (Mori and Ohshima, 1995). unc-86 encodes a POU-domain transcription factor (Finney, et al, 1988) that has two independent helix-turn-helix DNA binding domains (Klemm et al, 1994). UNC-86 accumulates in the nuclei of 47 neuronal lineages in wild-type animals, including all cells affected by unc-86 mutations (Finney and Ruvkun, 1990). The 57 postmitotic neurons that express UNC- 86 constitute 27 different types of neurons, including interneurons, sensory neurons and motor neurons, many of which are defective in unc-86 mutants. Genes regulated by unc-86 that act in just one of these neural classes have been detected. The LIM-homeobox gene mec-3 (Way and Chalfie, 1988) is necessary for the differentiation of the six mechanosensory neurons, and mec-7 encodes a mechanosensory neuron-specific β-tubulin that is necessary for the mechanosensation (Savage et al., 1989). The generation of the mechanosensory neurons depends on unc-86 gene activity, and in wild-type UNC-86

2 1160 J. Y. Sze, Y. Liu and G. Ruvkun continues to be expressed in these neurons. UNC-86 has been shown to bind in vitro to the promoter regions of both mec-3 and mec-7 (Xue et al., 1992, 1993; Lichtsteiner and Tjian, 1995; A. Duggan and M. Chalfie, personal communication). Because many neural types specified by unc-86 execute distinct patterns of neural pathfinding and synaptic connectivities, UNC-86 may regulate the expression of distinct repertoires of downstream genes that actually mediate the differentiated features of each neuron class. Here we report the development of a strategy to identify the genetic cascades downstream of unc-86. Since unc-86 encodes a transcription factor, we reasoned that if unc-86 gene activity could be hyperactivated, it may increase or constitutively activate the expression of the genes normally regulated by it, and that such elevated expression of downstream genes might confer phenotypes in wild type or in animals bearing mutations in those downstream genes. This is conceptually similar to the use of activated ras and heterotrimeric G proteins in elucidation of downstream signal transduction pathways (Gaul et al., 1993; Wu and Han, 1994; Lackner et al., 1994; Mendel et al., 1995). To activate unc-86, we inserted the potent VP16 transactivation domain (Triezenberg et at., 1988; Aoyama et al., 1995) into the otherwise wild-type unc-86 genomic sequence. We find that UNC-86/VP16 is expressed in the correct neurons and can rescue the behavioral defects of unc-86 null mutants. We present molecular and genetic evidence that unc-86/vp16 is capable of hyperactivating the expression of mechanosensory neuron downstream genes mec-3 and mec-7, and that this activation of downstream gene expression is sufficient to suppress non-null mutations in these genes. We also find that unc-86/vp16 causes neural migration and axon outgrowth defects, suggesting that unc-86 regulates the expression of downstream genes that mediate these neural functions as well. MATERIALS AND METHODS Genetics The following strains were used: wild-type N2 (Brenner, 1974); the unc-86 alleles, n846, e1416 (Finney et al., 1988); the mec-3 alleles e1338 (Xue et al., 1993), u298, u312 (Way and Chalfie, 1989); the mec-7 alleles u305, u431, u88, e1506 (Savage et al., 1994); lin-15 (n765ts); and egl-1(n987). Construction of the unc-86/vp16 fusion gene is located on a 17 kb EagI fragment of the cosmid C30A5 that was cloned in pbluescript SK. The coding sequence of VP16 transactivation domain (amino acids 413 to 482) (Triezenberg et al., 1988) was amplified from the plasmid pbxgal-vp (R. Kingston) using the PCR primers CGCGGATCCAAGGAGCCCCCCCGACC- GATGTCAGC and CGGATCCTTGGGCATCGGTAAACATCT- GCTC. unc-86/vp16 was constructed by ligating this VP16 domain into the unique StyI site of, which places the domain 72 amino acids N-terminal to the unc-86 POU domain. To construct unc- 86/VP16( -POU), a 4.6 kb BclI fragment, which encompasses the entire unc-86 POU domain was removed from unc-86/vp16, and a DNA fragment which contains the SV40 nuclear localization sequence (Kalderon et al., 1984), the GFP (Aequorea victoria green fluorescent protein) coding region (Chalfie et al., 1994), and the unc untranslated region was inserted. Cellular and behavioral analyses unc-86 constructs were co-injected with the marker plasmids as described (Mello et al., 1991). In each case, the DNA concentration of the unc-86 plasmid was 20 ng/µl. Where indicated, mec-7/gfp or unc-86/gfp (Baumeister et al., 1996) was co-injected at the concentration of 50 ng/µl and 40 ng/µl, respectively. Between 6 to 21 transgenic lines were used for behavioral and mec-7/gfp expression analysis. Some experiments used an unc-86/vp16 fusion transgene that was stably integrated into the chromosome IV by gamma irradiation. UNC-86 and MEC-7 expression was detected by indirect immunofluorescence using anti-unc-86 antibodies and anti-mec-7 antibodies (M. Hamelin) as described by Finney and Ruvkun (1990). Neurons that express UNC-86 were identified on the basis of the position of their nuclei with respect to DAPI staining according to White et al. (1986). The neurons that express MEC-7 or mec-7/gfp were assigned on the basis of their positions and axonal morphology using DIC and fluorescent microscopy. The ectopic expression of mec-7/gfp in the HSN neurons was supported by the disappearance of this ectopic expression in egl-1(n987) which causes HSN-specific cell death (Desai and Horvitz, 1989). The behavioral assay for sensitivity to light mechanical stimulus was described by Chalfie and Sulston (1981). L4 larvae to young adults from multiple transgenic lines were tested. RESULTS AND DISCUSSION Design of the experiments The transcriptional activation function of many transcription factors, including members of the POU superfamily, is mediated by modular transcriptional activation domains whose activities can in turn be regulated, for example by phosphorylation (Tanaka and Herr, 1990; Yin et al., 1995). A 70 amino acid motif of the Herpes virus protein VP16 has been shown to confer potent transcriptional activation on a wide variety of transcription factors, both in vitro and in vivo, in organisms as diverse as yeast and mouse (Sadowski et al., 1988; Nevins, 1991). This transcription activation domain is thought to interact directly with components of the transcription machinery (Goodrich et al. 1993), including RNA polymerase itself (Xiao et al., 1994), to trigger transcription when it is brought to a promoter by a DNA binding domain. We reasoned that fusion of the VP16 activation domain to a wildtype unc-86 gene bearing a functional POU DNA binding domain (Tanaka and Herr, 1990) would bypass normal regulation of UNC-86 transcriptional activation function to express unc-86-regulated downstream genes at a higher than normal level. In addition, activated UNC-86/VP16 might bypass normal combinatorial control mechanisms so that unc-86- regulated genes, such as mec-3 or mec-7 that are normally expressed in only one of the 27 classes of unc-86-expressing neurons, might be ectopically expressed in the other neuron classes. This hyperactivation or promiscuous expression of downstream genes was expected to have genetic consequences that could be used to detect unc-86 gene activities in neural development and function, and to identify by genetic suppression screens the downstream genes that mediate these functions. The activated unc-86/vp16 gene and two control genes used in this study are described in Fig. 1. The genomic unc-86 region used in these constructs contains about 8 kb upstream of the transcription start, the entire transcribed region and about 5 kb downstream the putative poly-a site. This 17 kb genomic

3 VP16-activation of C. elegans UNC Fig. 1. Schematic representation of and unc-86/vp16 fusion genes. contains 17.3 kb of genomic sequence that complements unc-86 null alleles. The unc-86 transcription start is marked with an arrow and exons are shown in grey. The unc-86 POU specific domain and POU homeodomain are indicated. unc-86/vp16 has the VP16 transactivation sequence inserted inframe at the StyI site upstream of the unc-86 POU domain. unc-86/vp16 ( -POU) was derived from unc-86/vp16 by deleting the unc-86 coding region and 3 UTR from a region 63 bp downstream the StyI site, and replacing it with a DNA fragment containing SV40 nuclear localization sequence, the GFP coding sequence and unc-54 3 UTR. unc-86 promoter unc-86/ VP16 unc-86 promoter VP16 POUs POUh POUs POUh fragment is sufficient to direct a correct expression of UNC- 86 and rescues unc-86 null mutations (Finney and Ruvkun, 1990; Baumeister et al, 1996; this work). The unc-86/vp16 fusion gene was constructed by insertion of the 70 amino acid VP16 transactivation domain (Triezenberg et al., 1988) inframe 72 amino acids N-terminal to the beginning of the POU domain. The activities of VP16-activated unc-86 transgenes were compared to control transgenes for the ability to complement an unc-86 null mutant, to suppress mec- 3 and mec-7 mutants, and to confer other neural phenotypes. To establish whether the phenotypes conferred by unc- 86/VP16 are based on binding to its normal downstream gene targets, the activities of a control gene with a deletion of the POU DNA binding domain, unc- 86/VP16( -POU), was compared to unc-86/vp16 and transgenes. Multiple transgenic C. elegans strains carrying each fusion gene were analyzed. unc-86/ VP16( -POU) unc-86 promoter VP16 GFP unc-86/vp16 rescues the mechanosensation defects of unc-86 null mutants unc-86 loss-of-function mutants have neuroblast cell lineage defects that result in failure to generate the six touch receptor NLS 200bp Fig. 2. unc-86/vp16 complements the developmental and functional defects of unc-86 null mutants. (A- C) Immunostaining with anti-unc- 86 antibody. Animals are L4 larvae. (A) Wild type, showing UNC-86 expression in the hermaphroditespecific neuron (HSN) and the mechanosensory neurons PVM and PVD. (B) unc-86(n846) null mutant, showing no UNC-86. (C) unc- 86(n846); Ex(unc-86/VP16) transgenic animals express UNC- 86/VP16 in neurons that normally express UNC-86. (D-G) Immunostaining with anti-mec-7 antibody. All the animals are young adults. (D) Wild type, showing the mechanosensory neuron ALM and PLM axons. (E) unc-86(n846) mutants alter the lineages of the neuroblasts that normally generate the mechanosensory neurons and thus fail to express MEC-7. (F-G) unc-86(n846); Ex(unc-86/VP16) animals restore the ability to express MEC-7 in the mechanosensory neurons. Cell migration and axonal defects were observed in some of the transgenic animals (see text); the neurons with correct morphology are shown. (H) unc-86/vp16 rescues mechanosensitivity of an unc-86(n846) mutant. The animals were tapped alternately at the head and tail regions as described by Chalfie and Sulston (1981). The percentage of trials that an animal responded to are shown. Number of animals scored for each strain are: wild type, 9; unc-86(n846), 9; unc-86(n846); Ex(), 10; unc- 86(n846); Ex(unc-86/VP16), 10; unc-86(n846); Ex(unc-86/VP16( - POU)), 33. Error bars represent the standard error of the mean. H Response to light mechanical stimulus (%) wild type no transgene Ex Ex unc-86/ VP16 Ex unc-86/ VP16( -POU) unc-86(n846)

4 1162 J. Y. Sze, Y. Liu and G. Ruvkun neurons ALML, ALMR, PLML, PLMR, AVM, and PVM (Chalfie and Sulston, 1981; Chalfie et al., 1981). Because of these missing mechanosensory neurons, the animals are mechanosensation defective (Mec). UNC-86 protein is expressed in the mechanosensory neurons as well as all other neurons that normally express UNC-86 in unc-86 null mutant animals carrying extrachromosomal or unc- 86/VP16 transgenes, whereas mechanosensory neurons as well as many other neurons are missing in unc-86 mutant animals carrying extrachromosomal unc-86/vp16( -POU) or no transgene (Fig. 2). Activated unc-86/vp16 as well as unc- 86(+) rescues the Mec phenotype of animals bearing chromosomal unc-86 null mutations e1416 or n846 (Fig. 2H), even though axonal defects and novel behavioral abnormalities are observed in some animals carrying unc-86/vp16 (see below). Consistent with the rescue of these behavioral phenotypes, the mechanosensory neuron-specific gene mec-7 is expressed in transgenic unc-86 mutant animals carrying either or unc-86/vp16 transgenes but not in unc-86 mutants bearing an unc-86/vp16( -POU) transgene or no transgene (Fig. 2). Although UNC-86/VP16( -POU) is expressed in the many neural cell lineages that normally express UNC-86 and is localized to the nucleus, the gene is inactive for all UNC-86/VP16 activities. It does not rescue the mechanosensation defects of an unc-86 null mutant (Fig. 2H), nor does it confer the abnormal axonal morphology and behaviors caused by unc-86/vp16 (see below). These data suggest that the neural activities of UNC- 86/VP16 depend on POU-domain mediated DNA binding of this activated transcription factor to downstream targets. unc-86/vp16 suppresses non-null mutations in mec- 3 and mec-7 One prediction from studies of VP16 activation domain fusions to transcription factors (Sadowski et al., 1988) is that unc- 86/VP16 may increase or cause constitutive expression of UNC-86 target genes. We reasoned that such increased expression of downstream genes might suppress weak but not null mutations in the downstream genes because an increased expression of partially active product of these mutant genes may supply sufficient gene activity. We used the probable downstream genes, mec-3 and mec-7 to test this approach. mec- 7 encodes a mechanosensory neuron-specific tubulin that is necessary for mechanical signal transduction (Savage et al., 1989; Hamelin et al., 1992). mec-3 encodes a LIM-homeodomain protein that is expressed in mechanosensory neurons (Way and Chalfie, 1989). In mec-3 loss-of-function mutants, these neurons are formed, but have no mechanosensory neuron characteristics: for example, mec-7 is not expressed and the animals are mechanosensory defective (Chalfie and Sulston, 1981; Hamelin et al., 1992). unc-86/vp16 suppresses non-null mutations in mec-3. We compared the effect of unc-86/vp16 on three mec-3 alleles, u298, u312 and e1338 (Fig. 3A; Table 1). All of these mec-3 alleles cause a 90-95% penetrant mechanosensory defective phenotype and significantly reduce the expression of MEC-7 (Fig. 3A; Table 1). mec-3(u298) and mec-3(u312) animals carrying unc-86/vp16 are nearly wild type in response to touch compared to the parent mec-3 mutant strains (Fig. 3A). This suppression is dependent on the VP16 activation domain, because transgenic does not suppress these mec-3 alleles, and it is dependent on the POU DNA-binding domain, because unc- 86/VP16( -POU) does not suppress these mec-3 alleles (Fig. 3A). unc-86/vp16 does not suppress the mechanosensation defects of the mec-3(e1338) null allele (Fig. 3A). The improved mechanosensation in mec-3 mutant animals bearing unc-86/vp16 is associated with MEC-7 expression, as determined by mec-7/gfp expression analysis (Table 1) and anti-mec-7 antibody staining (Fig. 3C). mec-7/gfp expression was observed at much higher frequency in the transgenic mec-3(u298) and mec-3(u312) bearing unc-86/vp16 than in mec-3(u298) or mec-3(u312) animals bearing unc- 86(+) or unc-86/vp16( -POU) transgenes (Table 1). The level of mec-7 expression appeared to be higher in the mec-3(u312); Ex unc-86/vp16 animals than in the mec-3(u298); Ex unc- 86/VP16 animals (not shown), as was the genetic suppression of mechanosensory behavior defects (Fig. 3A). unc-86/vp16 does not activate mec-7 expression in mec-3(e1338). mec-3(e1338) is a W69amber mutation that truncates the MEC-3 LIM domains and homeodomain, and thus is likely to be a null allele (Xue et al., 1993; Fig. 3A). Consistent with no mec-3 gene activity from this allele, hyperactivation by unc- 86/VP16 does not suppress the Mec mutant phenotype. mec- 3(u298) is a transposon Tc1 insertion in the mec-3 promoter (Way and Chalfie, 1988; Xue et al., 1992; Fig. 3A). The mec- 3(u298) Tc1 insertion separates the distal UNC-86 binding site from two proximal UNC-86 binding sites by 1.6 kb, which may affect cooperative DNA binding or cooperative transcriptional activation by UNC-86 (Xue et al., 1992). The suppression of mec-3(u298) by unc-86/vp16 but not suggests that increased activation function of UNC-86/VP16 can activate MEC-3 expression from the enfeebled mec-3(u298) promoter that is not responsive to UNC-86. This suppression is still weaker than that observed with mec-3(u312), suggesting that UNC-86/VP16 also activates mec-3 expression via the three UNC-86 binding sites in the mec-3 promoter. While the mec- 3(u312) molecular lesion is not known, the suppression of this allele by unc-86/vp16 suggests that the mec-3 gene or gene product is partially active in this mutant. unc-86/vp16 also suppresses non-null mutations in mec-7. The mec-7 mutants u431, u88, and u305 are 80% to 90% mechanosensory defective (Savage et al., 1989; Fig. 3B). unc- 86/VP16 strongly suppresses the mechanosensory defects of mec-7(u305) and mec-7(u88) but does not suppress mec- 7(u431) (Fig. 3B). This suppression is dependent on the VP16 activation domain and the POU DNA-binding domain because and unc-86/vp16( -POU) do not suppress these mec-7 alleles (Fig. 3B). The molecular lesions of these mec-7 mutant alleles and their alignment with tubulin functional domains (Savage et al., 1994) can be easily reconciled with this pattern of genetic suppression (Fig. 3B). u305 is a missense (G34S) mutation in a non-conserved region, whereas u431 is a nonsense mutation (Q280Amber) which is expected to truncate the MEC-7 protein upstream of putative assembly, dimerization, and MAP-binding regions, and u88 is a nonsense mutation (R318Opal) which is expected to truncate only the dimerization and MAP-binding regions. The nonsense allele mec-7(u88) R318opal that is suppressed by unc-86/vp16 is further C terminal and thus truncates less of the MEC-7 protein than mec-7(u431) Q280amber that is not suppressed. These data suggest that increased or constitutive expression of the mutant MEC-7G34S or MEC-7R318Stop can compensate for their deficits in mec-7 gene activity; whereas increased or con-

5 VP16-activation of C. elegans UNC Table 1. unc-86/vp16 suppresses mechanosensory neuron differentiation defects in non-null mec-3 mutants Percentage of cells that express mec-7/gfp Other Number Relevant nerve ring of genotype* FLP AVM ALM PVD PVM PLM ALN HSN PHC BAG neurons animals Wild-type Wild-type; Ex() Wild-type; Ex(unc-86/VP16) mec-3 null mutant: mec-3(e1338); Ex(unc-86/VP16) mec-3 weak mutant: mec-3(u312) nd mec-3(u312); Ex(unc-86/VP16) nd mec-3(u312); Ex() nd mec-3(u312); unc-86(n846); nd Ex(unc-86/VP16) mec-3(u312); unc-86(n846); nd Ex(unc-86 (+)) mec-3(u298); Ex() nd mec-3(u298); Ex(unc-86/VP16) nd *Transgenic animals carry a mec-7/gfp reporter gene. Numbers listed are the percentage of cells that express mec-7/gfp. Most of the neurons are bilaterally symmetric, except AVM and PVM. The mechanosensory neurons that normally express MEC-7 are in bold type. Ectopic expression of mec-7/gfp in 4-6 neurons around the nerve ring was predominantly observed in L1-L2 larvae animals. These neurons have not been unambiguously identified and they were not observed with anti-mec-7 antibody. FLP, PVD, ALN and BAG express mec-7/gfp and mec-7/lacz in wild-type animals (Hamelin et al., 1992); sem-4 mutations also cause ectopic expression of MEC-7 in PHC (Mitani et al.,1993; Basson and Horvitz, 1996). mec-7/gfp is expressed HSN only in the animals carrying unc-86/vp16. The additional mec- 7/GFP expressing neurons were confirmed to be HSNs, because the ectopic expression was absent in egl-1(n986) which causes HSN-specific cell death (Desai and Horvitz, 1989). stitutive expression of the MEC-7Q280Stop, which presumably cannot be assembled into the microtubules (Savage et al., 1994), cannot supply sufficient MEC-7 activity for normal mechanosensation. As determined by visual inspection of GFP fluorescence, expression of mec-7 is slightly increased in animals bearing unc-86/vp16 relative to animals carrying. We observe no detectable increase in MEC-7 abundance nor ectopic expression of MEC-7 in the other 47 neurons that express UNC-86/VP16, as determined by immunofluorescence (Fig. 2). This suggests that the novel genetic suppression activities of unc-86/vp16 are due to a moderate increase in downstream gene expression. From these experiments we conclude that the ability of UNC-86/VP16 to suppress mec-3 or mec-7 mutations is dependent on the VP16 activation domain, the UNC-86 POU DNA binding domain, as well as partial mec-3 or mec-7 gene activities. In addition, because unc-86/vp16 does not suppress the null allele mec-3(e1338), the strong activation function of UNC-86/VP16 cannot bypass the requirement for MEC-3 in the differentiation of the mechanosensory neuron. In fact, in the mec-3(e1338) mutant, mechanosensory neurons visualized with an unc-86/gfp fusion gene (Baumeister et al., 1996) have severely truncated or no process, and unc-86/vp16 does not suppress this defect (data not shown). The MEC-3 requirement for UNC-86 and UNC-86/VP16 function in mechanosensory neuron differentiation is consistent with DNA binding studies which show that MEC-3 is a combinatorial partner for UNC- 86 DNA binding (Xue et al., 1992, 1993; A. Duggan and M. Chalfie, personal communication; Lichtsteiner and Tjian, 1995). An increase in MEC-3 level as well as the increased activation function of UNC-86/VP16 may both contribute to the suppression of weak mec-7 mutations by unc-86/vp16. Another possibility is that UNC-86/VP16 may increase the expression of an unknown transcription factor that in turn increases the expression of both mec-3 and mec-7. The observations that mec-3 and mec-7 expression depend on unc-86 gene activity, that UNC-86 continues to be expressed in mature mechanosensory neurons (Finney and Ruvkun, 1990; Hamelin et al., 1992), and that UNC-86 binds directly to the mec-7 promoter (A. Duggan and M. Chalfie, unpublished results) support a model of direct regulation. unc-86/vp16 suppression of the weak mec-3 or mec-7 alleles is neither dependent on nor affected by the presence of wild-type UNC-86 from chromosomal (Fig. 3AB, Table 1). Thus VP16 activation of UNC-86 is genetically dominant. Transgenic at a similar gene dosage does not show any of the dominant gene activities shown by unc- 86/VP16. However, unc-86/vp16 displays normal unc-86 gene activity: it complements an unc-86 null mutant as well as unc- 86(+) (Fig. 2). These data argue that unc-86/vp16 possesses both wild-type unc-86 gene activity as well as a novel dominant genetic activity that suppresses weak mutations in downstream genes. The dominance of unc-86/vp16 favors the model that it activates the expression of downstream genes much more potently than. unc-86/vp16 perturbs neural migration and pathfinding Because unc-86 expression is activated early in neural differentiation (Finney and Ruvkun, 1990), we investigated whether the VP16-mediated activation of unc-86 downstream genes causes defects in early neurogenic events such as neural migration and pathfinding. Using mec-7/gfp to reveal the

6 1164 J. Y. Sze, Y. Liu and G. Ruvkun A Response to light mechanical stimulus(%) 100 no transgene Ex unc-86 (+) wild type mec-3(u312); mec-3(u312); unc-86(n846) Ex unc-86/vp16 Ex unc-86/vp16( -POU) mec-3(u298); mec-3(e1338); mec-3(e1338); unc-86(n846) B Response to light mechanical stimulus (%) wild type mec-7(u305); mec-7(u305); unc-86(n846) no transgene Ex unc-86 (+) Ex unc-86/vp16 Ex unc-86/vp16( -POU) mec-7(u431); mec-7(u88); mec-3 mec-7 u298 (Tc1) e1338 (W69amber) u305 (G34S) u431(q280amber) u88 (R318opal) LIM-domain homeodomain 200 bp 200 bp Fig. 3. unc-86/vp16 suppresses non-null mutations in downstream genes mec-3 and mec-7. (A) unc-86/vp16 suppresses weak but not null mutations in the LIM-homeobox gene mec-3. Top panel: the percentage of trials that animals responded to light touch. Number of animals scored for each strain are: wild type, 9; mec-3(u312), 11; mec-3(u312); Ex(), 30; mec-3(u312); Ex(unc-86/VP16), 10, mec-3(u312); Ex(unc-86/VP16( -POU) 38; unc-86(n846); mec- 3(u312), 21; unc-86; mec-3(u312); Ex(), 59; unc-86(n846); mec-3(u312); Ex(unc-86/VP16), 58, mec-3(u298), 45; mec-3(u298); Ex(), 32; mec-3(u298); Ex(unc-86/VP16), 60; mec- 3(u298); Ex(unc-86/VP16( -POU)), 61; mec-3(e1338), 22, mec- 3(e1338); Ex(unc-86/VP16), 89, unc-86(n846); mec-3(e1338); Ex(), 48; unc-86(n846); mec-3(e1338); Ex(unc-86/VP16), 38. Error bars represent the standard error of the mean. Bottom panel: a schematic representation of the mec-3 gene. The regions encoding the LIM-domain and homeodomain are indicated, and the UNC-86 and MEC-3 binding sites (Xue et al., 1992) are representated as circles containing 86 and rectangular boxes containing 3, respectively. The molecular lesions of the alleles u298 and e1338 are indicated (Xue et al., 1993), and the molecular lesion of the allele u312 is not known. (B) unc-86/vp16 suppresses weak mutations in the mechanosensory neuron-specific tubulin gene mec-7. Top panel: the percentage of trials that animals responded to light mechanical stimuli. Number of animals scored for each strain are: wild type, 9; mec-7(u305), 19; mec-7(u305); Ex(), 31; mec-7(u305); Ex(unc-86/VP16), 44; mec-7(u305); Ex(unc-86/VP16( -POU)), 28; unc- 86(n846); mec-7(u305), 21; unc-86(n846); mec-7(u305); Ex(), 55; unc-86(n846); mec-7(u305); Ex(unc-86/VP16); 54; mec-7(u431), 22; mec-7(u431); Ex(unc-86/VP16), 19; mec-7(u88), 13; mec-7(u88); Ex(), 44; mec-7(u88); Ex(unc-86/VP16), 34; mec-7(u88); Ex(unc-86/VP16( -POU)), 45. Error bars represent the standard error of the mean. Bottom panel: a schematic representation of the mec-7 β- tubulin gene. The transcription start is indicated by an arrow; exons are boxed, and the UNC-86 and MEC-3 binding sites (Duggan and Chalfie, unpublished) are indicated as circles containing 86 and rectangular boxes containing 3, respectively. The mec-7 alleles and their molecular lesions are indicated. The mec-7 gene structure is adapted from Savage et al. (1994). (C-D) Immunostaining with anti-mec-7 antibody. All the animals are L4 larvae. Anterior is to the left and dorsal is on the top. (C) mec-3(u312), the expression of MEC-7 is greatly reduced. (D) mec- 3(u312); Ex(unc-86/VP16) enhances the expression of MEC-7 in the PVM neuron. The arrowhead indicates an extra posteriorly directed axon, which is often observed in unc-86/vp16 transgenic animals. positions of cell bodies and axons of the mechanosensory neurons, we find that activated UNC-86/VP16 causes defects in the migration and axon outgrowth (Table 2, Fig. 4). unc- 86/VP16 causes mechanosensory neuron migration defects predominantly in the anterior body region (Fig. 4E, Table 2). Other common defects are sprouting of extra short branches from cell bodies and processes (Fig. 4BF), extra posteriorly directed axons (Fig. 4E), and abnormal varicosities (Fig. 4F). Less frequently, there are striking defects in pathfinding. For example, axon processes initially navigate in the wrong direction, but eventually return to the normal path (Fig. 4B), or an additional axon sprouts from the cell body and follows an

7 VP16-activation of C. elegans UNC Table 2. unc-86/vp16 causes neuron migration, axonal outgrowth and pathfinding defects % cell migration defect % axonal outgrowth defect % pathfinding defect Relevant genotype* ALM AVM PVM PLM ALM AVM PVM PLM ALM AVM PVM PLM ; Ex() ; Ex(unc-86/VP16) unc-86(n846); Ex() unc-86(n846); Ex(unc-86/VP16) mec-7(e1506); ; Ex(unc-86/VP16) *Transgenic animals carry the mec-7/gfp reporter gene to reveal mechanosensory neuron anatomy. Positions of mechanosensory neuron cell bodies and axonal processes were revealed by GFP fluorescence. mec-7(e1506) is a probable mec-7 null mutant (Savage et al., 1994). lin-15 and rol-6 were used as transformation markers in and unc-86(n846) transgenic animals, respectively. For each neuron type, the neuroanatomy of animals was examined. opposite path from the normal process (Fig. 4C). Although abnormal axon outgrowth is occasionally observed in unc- 86(+) transgenic animals, unc-86/vp16 transgenic animals show more dramatic defects at much higher frequencies (Table 2). These unc-86/vp16 activities are independent of unc- 86(+), showing that they are genetically dominant (Table 2). The defects in mechanosensory neural development are also independent of mec-7 gene activity, showing that these migration and pathfinding defects are not caused by increased expression of this tubulin gene (Table 2). Regulated expression of neural growth associated proteins (Aigner et al., 1995), cell adhesion molecules (Hynes and Lander, 1992), and surface receptors for extracelluar cues (Culotti, 1994) may be necessary for neural migration and pathfinding (Goodman and Shatz, 1993). These molecules decorate particular sets of axons, and even particular regions of axons to attract or repel particular other neurites (Kolodkin et al., 1992; Meier et al., 1993). In order to express such a spectrum of surface adhesion and receptor molecules, the pattern of gene expression in the developing neuron must be dynamic so that a series of surface receptors are expressed over time to decorate a series of segments of a developing neurite. unc- 86/VP16 may bypass such detailed modulation of its transcriptional activity and instead express downstream receptor and adhesion genes constitutively thus perturbing neural pathfinding. For example, ectopic expression of UNC-5, the putative receptor for the C. elegans netrin UNC-6 (Hedgecock et al., 1990; Ishii et al., 1992) in the mechanosensory neurons redirects their axon trajectories (Hamelin et al., 1993). UNC-86/VP16 dependent axon sprouting was observed predominantly in adult animals (data not shown), suggesting that the mechanosensory neuron anatomy remains plastic at late developmental stages. unc-86 continues to be expressed in the mature nervous system (Finney and Ruvkun, 1990), suggesting that it may continue to regulate the expression of genes that maintain neural function. Constitutively active UNC-86/VP16 may be decoupled from normal regulation of UNC-86 activity, perhaps by neural activity (Goodman and Shatz, 1993, Bourtchuladze et al., 1994; Yin et al., 1995), so that it may constitutively activate expression of proteins that mediate synaptic signaling, thus causing the late onset neural outgrowth defects we observe. Interestingly, the unc-86 mammalian orthologue Brn-3.0 regulates the Fig. 4. unc-86/vp16 causes cell migration and axonal outgrowth defects. Anterior is to the left and dorsal is on the top in all panels. (A) Wild-type PVM in an transgenic animal. A single process enters the ventral cord by a commissure and runs anteriorly along the ventral cord (White et al., 1986). (B-C) unc- 86/VP16 alters the axonal morphology of PVM; the process often runs posteriorly first and then turns anteriorly (B) or simply runs laterally (C). Supernumerary processes (arrow) often sprout from the main process (B) or from the cell body (C). (D) Wild-type ALM in an transgenic animal: the cell body is situated more posterior than AVM and a single axon runs anteriorly. (E) The cell body of ALM in unc- 86/VP16 transgenic animals is shifted to a position more anterior than AVM, and it has an unusual long posterior process. (F) unc-86/vp16 animals have abnormal varicosities along the ALM process and extra branches (arrow) sprout from the process and cell body. All strains carry the lin-15(n765ts) mutation and a lin-15(+) co-injection marker. The transgenic animals also carry the mec-7/gfp reporter gene to visualize mechanosensory neuroanatomy.

8 1166 J. Y. Sze, Y. Liu and G. Ruvkun neurite outgrowth in conjunction with a downstream synaptic signaling regulatory molecule SNAP-25 (Lakin et al., 1995). Other phenotypes also support the model that synaptic signaling is intensified by activated unc-86/vp16. Even with the defects in neural development, animals bearing unc-86/vp16 can respond to light mechanical touch. This implies that the touch receptor neurons form sufficient synapses and gap junctions with the command interneurons AVA, AVB, AVD, PVC, and LUA that initiate forward and reverse movements in response to mechanical stimuli (Chalfie et al., 1985; Maricq et al., 1995; Hart et al., 1995). However, although unc-86/vp16 animals respond to touch, their response to repetitive touches is abnormal. Wild-type animals respond to multiple light touches about 15 times before that response habituates (Rankin et al., 1990). After habituation to touch, the animals continue to move but do not reverse direction of movement in response to touch. Wild type can be touched more than 100 times and continue to move. Animals bearing the unc-86/vp16 transgene also habituate to touch; but in contrast to wild type, unc-86/vp16 animals become immobilized after as few as 8 touches (average = 49). This unc-86/vp16 activity is dominant to. The unc-86/vp16 induced immobilization is dependent on mechanosensory neurons because it does not occur in a mec-3 mutant background but is not dependent on mec-7 mediated mechanosensation because it occurs even in the mec-7(u431) mutant (data not shown). Because all movement is disabled in unc-86/vp16 animals after a series of light touches, it is likely that the command interneurons that also mediate stimulation from other sensory modalities are incapacitated by excessive signaling from the mechanosensory neurons. UNC-86 expression in the mature nervous system (Finney and Ruvkun, 1990) may normally regulate the expression of genes that mediate synaptic transmission and signal transduction. For example, unc-86 is necessary for serotonin accumulation in the HSN neuron (Desai et al., 1988). Increased or ectopic expression by unc-86/vp16 of analogous synaptic signaling genes in the mechanosensory neurons could increase the number of signals or intensity of signaling from synapses or gap junctions, and thus saturate the postsynaptic signaling pathways of the command interneurons. unc-86/vp16 causes serotonergic defects and ectopic expression of mec-7 in the HSN neurons In contrast to its action in the mechanosensory neurons, unc- 86/VP16 does not supply gene activity in the serotonergic HSN motor neurons that stimulate egg-laying (Desai et al., 1988). unc-86/vp16 does not rescue the defects in serotonin synthesis and egg-laying behavior of an unc-86 null mutant, unlike an transgene (data not shown). Even wild-type animals carrying unc-86/vp16 are egg-laying defective (Egl) and do not accumulate serotonin (data not shown), showing that unc-86/vp16 is dominant for this phenotype. The similarity of the unc-86 loss-of-function and UNC-86/VP16 dominant phenotypes in the HSN suggests that UNC-86 may normally repress the activity of genes that downregulate serotonin synthesis or upregulate serotonin release, so that the consequences of loss of wild-type UNC-86 or the presence of UNC-86/VP16 would be the same: decreased serotonin levels. Alternatively, UNC-86/VP16 may upregulate the expression of proteins that mediate the secretion of serotonin, thus decreasing serotonin levels in the HSN. UNC-86/VP16 also causes ectopic mec-7 expression in the HSN neurons (Table 1). While the expression of mec-7 in the HSN suggests a transformation of the HSN towards mechanosensory neural fate, other HSN fates remain. For example, the neurons show an HSN-like process anatomy and die in the egl-1 mutant (Table 1). At lower frequency, UNC- 86/VP16 also induces ectopic mec-7/gfp expression in 6 8 nerve ring neurons and in two tail neurons (Table 1). Ectopic expression of mec-7 in the HSN as well as in the other neurons is dependent on mec-3 gene activity (Table 1). None of these neurons express MEC-3 in wild-type animals (Way and Chalfie, 1989), suggesting that unc-86/vp16 induces promiscuous mec-3 expression in a subset of the unc-86 expression pattern. Analogous ectopic mec-7 expression in sem-4, egl-44, and egl-46 mutants has been noted but not in the HSN (Mitani et al., 1993; Basson and Horvitz, 1996); these genes may encode negative regulators of mec-3 expression or activity in other neurons similar to an HSN negative regulator whose function is bypassed by UNC-86/VP16. Misexpression of mec-3 in the HSN neuron is not the cause of the egg-laying defects of unc-86/vp16 animals. The downregulation of HSN serotonin levels as well as the Egl phenotype induced by unc-86/vp16 is not dependent on mec- 3 gene activity (data not shown), suggesting dysregulated expression of other downstream genes causes the HSN defects in the serotoninergic signaling. Concluding remarks Genetic suppression of activated regulatory genes has been a powerful tool in the identification of downstream genes in genetic pathways (Gaul et al., 1993; Wu and Han, 1994; Lackner et al., 1994). For example, some suppressors of hyperactive ras mutations have no phenotype alone and thus would have been missed in genetic screens for mutants with phenotypes similar to ras loss-of-function mutants (Lackner et al., 1994). We suggest that an activated unc-86 gene can be used analogously for pathway analysis in this transcriptional regulatory cascade. This activated transcription factor has two classes of effects: (1) it suppresses non-null mutations in downstream genes; (2) it causes novel neural developmental and behavioral phenotypes in wild type that may be due to increased or promiscuous expression of downstream genes. While definitive genetic proof that particular transcription factors directly regulate cognate downstream genes has been achieved with compensatory changes in homeodomain binding site specificity and DNA binding sites (Schier and Gehring, 1992), the simplicity of genetic suppression by a VP16 fusion gene provides a practical means of identifying unknown downstream genes in existing mutant collections or in specific genetic screens. This approach could be generally useful in the analysis of regulatory pathways downstream of transcriptional factors, such as the genes downstream of CREB that actually mediate the neural plasticity regulated by this molecule (Bourtchuladze et al., 1994; Yin et al., 1995). Given the potent suppression of non-null mutations in downstream genes by unc-86/vp16, genetic screens for mutations that are suppressed by VP16 fusion to transcription factors could identify bona fide downstream genes. For example, we are now testing whether unc-86/vp16 can suppress other mutants with phenotypes, such as egg-laying defects, thermotaxis defects, chemosensory defects, in common with unc-86 loss-offunction mutants. Suppression of the mutant phenotype of these

9 VP16-activation of C. elegans UNC candidates by unc-86/vp16 would strongly suggest that the particular gene is normally directly regulated by unc-86. In an analogous manner, the novel phenotypes conferred by fusion of VP16 to a transcription factor, for example in the case of unc- 86/VP16, neural pathfinding defects and serotonin-regulated egglaying defects, can be genetically suppressed to identify the downstream genes that mediate these phenotypes. Genes identified in such genetic suppression screens are not expected to be limited to bona fide downstream genes. While the suppression of weak mutations in the known downstream genes mec-3 and mec-7 shows that VP16 hyperactivates unc-86 gene activity to increase downstream gene expression, the ectopic mec-3 and mec-7 gene expression, and the neural pathfinding defects induced by unc-86/vp16 raise the spectre that neomorphic gene activities result from VP16 fusion. Neomorphic unc- 86/VP16 gene activities could be due to misexpression of bona fide downstream genes in incorrect neurons, as in the case of mec-3 and mec-7 in the HSN, or promiscuous expression of genes not normally regulated by unc-86. In the former case, genetic suppression of these phenotypes, for example the egglaying defects or the neural pathfinding defects, will identify true unc-86-regulated downstream genes, whereas in the latter case, such suppression will identify genes not in the unc-86 pathway whose promiscuous expression by unc-86/vp16 causes the phenotype. In addition, some mutations that are suppressed by UNC-86/VP16 may be in proteins that interact with the protein products of unc-86 regulated genes: UNC-86/VP16 activated increase in MEC-7 expression levels might suppress mutations in genes that encode proteins that normally interact with MEC- 7. This would be akin to high copy suppression in yeast (Reed et al., 1989). Finally, it is possible that unc-86/vp16 could increase the level of other transcription factors downstream that then indirectly suppress mutations in genes not directly regulated by unc-86. However, only if the level of such downstream transcription factors were also limiting would an increase in their level suppress mutations further down the cascade, whereas genes directly regulated by unc-86 would be more likely to become hyperactivated by the unc-86/vp16 fusion gene. Nevertheless, even if the downstream genes identified by VP16 genetic suppression are indirectly regulated by unc-86, they are still likely to function in the same genetic pathway being explored. In conclusion, these screens, like traditional genetic screens, could generate a mixture of mutations in true downstream genes and in adventitiously regulated genes. Secondary screens, for example, enhancement of unc-86 weak mutant phenotypes would distinguish the true downstream genes. 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