A universal transportin drives stochastic choice of olfactory neurons via specific nuclear import of a sox-2-activating factor

Amel Alqadaha, Yi-Wen Hsieha,1, Rui Xionga,1, Bluma J. Leschb, Chieh Changa, and Chiou-Fen Chuanga,2

aDepartment of Biological Sciences, University of Illinois at Chicago, IL 60607; and bDepartment of Genetics, Yale University School of Medicine, New Haven, CT 06510

Edited by Iva Greenwald, Columbia University, New York, NY, and approved October 31, 2019 (received for review June 25, 2019) Stochastic neuronal cell fate choice involving notch-independent mechanisms act downstream of the BK potassium channels to mechanisms is a poorly understood biological process. The induce the AWCON identity. Caenorhabditis elegans AWC olfactory neuron pair asymmetrically Here, we identify a role of the karyopherin imb-2/transportin 1 differentiates into the default AWCOFF and induced AWCON subtypes downstream of the SLO BK potassium channels in promoting in a stochastic manner. Stochastic choice of the AWCON subtype is the AWCON subtype from an unbiased forward genetic screen. established using gap junctions and SLO BK potassium channels to We show that asymmetrical expression of imb-2 in AWCON cells, repress a calcium-activated protein kinase pathway. However, it is which is dependent on nsy-5 (gap junction) and slo-1 (BK po- unknown how the potassium channel-repressed calcium signaling is tassium channel), is necessary and sufficient for AWC asymmetry. translated into the induction of the AWCON subtype. Here, we iden- In addition, IMB-2 localizes in close proximity to the homeo- tify a detailed working mechanism of how the homeodomain-like domain-like transcription factor NSY-7 and mediates nuclear transcription factor NSY-7, previously described as a repressor in the transport of NSY-7 to specify the AWCON subtype. Furthermore, maintenance of AWC asymmetry, couples SLO BK potassium chan- we reveal an activating function of NSY-7, which was previously nels to transactivation of sox-2 expression for the induction of the described as a repressor in the maintenance of AWC asymmetry ON AWC subtype through the identification of a unique imb-2 (trans- (14), in sox-2 expression by binding to its upstream regulatory portin 1) allele. imb-2 loss-of-function mutants are not viable; how- ON sequence to induce the AWC identity. Together, our study DEVELOPMENTAL BIOLOGY ever, we identify a viable imb-2 allelefromanunbiasedforward demonstrates that imb-2/transportin 1 functions to mediate nu- genetic screen that reveals a specific role of imb-2 in AWC olfactory clear transport of NSY-7 in AWC neurons, which, in turn, acti- neuron asymmetry. IMB-2 specifically drives nuclear import of NSY- vates sox-2 expression to promote the AWCON subtype. 7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box transcription factor SOX-2 for the specifi- Results ON cation of the AWC subtype. This study provides mechanistic in- The vy10 Mutation Causes a Defect in AWC Asymmetry. Wild-type sight into how NSY-7 couples SLO BK potassium channels to animals have 1 AWCON subtype, expressing the G protein-coupled transactivation of sox-2 expression for the induction of the receptor (GPCR) str-2,andoneAWCOFF subtype, expressing AWCON subtype. Our findings also provide structure-function in- sight into a conserved amino acid residue of transportins in brain Significance development and suggest its dysfunction may lead to human neurological disorders. Stochastic cell fate decisions are conserved and prominent pro- transportin 1 | stochastic choice | sox-2 | NSY-7 | asymmetry cesses during development, but the underlying molecular mechanisms are only partly understood. In the nematode Caenorhabditis elegans, the AWC sensory neuron pair asym- tochastic cell fate decisions are conserved and prominent metrically differentiates into 2 distinct identities in a stochastic Sprocesses during development, but the underlying molecular – manner. Through identification of a unique transportin allele, mechanisms are only partly understood (1 4). The Caenorhabditis we elucidate a mechanism by which a homeodomain-like fac- elegans (C. elegans) AWC pair of olfactory neurons acquires 2 ON OFF tor couples voltage- and calcium-activated potassium channels mutually exclusive subtypes (AWC and AWC ) and distinct to transactivation of a HMG-box transcription factor expression functions through a stochastic coordinated cell signaling event – for the stochastic choice of AWC identities. We show that (5 11), rendering it an excellent system to identify novel mo- transportin drives nuclear import of the homeodomain-like lecular mechanisms controlling stochastic cell fate specification. factor to activate the expression of the HMG-box transcription Stochastic choice of AWC olfactory neuron subtypes is estab- factor for stochastic AWC identities. Our findings also provide lished during late embryogenesis and maintained throughout structure-function insight into a conserved amino acid residue – OFF adulthood (12 15). The default AWC neuron is specified via of transportins in cell type diversification. a calcium-activated protein kinase pathway downstream of voltage- gated calcium channels (15, 16). In this pathway, the TIR-1 (Sarm1) Author contributions: A.A., Y.-W.H., R.X., B.J.L., C.C., and C.-F.C. designed research; A.A., adaptor protein assembles a synaptic calcium-signaling complex Y.-W.H., R.X., B.J.L., and C.-F.C. performed research; A.A., Y.-W.H., R.X., and C.-F.C. con- that consists of UNC-43 calcium/calmodulin-dependent protein tributed new reagents/analytic tools; A.A., Y.-W.H., R.X., C.C., and C.-F.C. analyzed data; kinase (CaMKII) and NSY-1 MAP kinase kinase kinase (ASK1 and A.A., Y.-W.H., R.X., B.J.L., C.C., and C.-F.C. wrote the paper. MAPKKK) in a microtubule-dependent manner (11, 12, 17, 18). The authors declare no competing interest. The NSY-5 gap junction protein innexin forms a transient gap This article is a PNAS Direct Submission. junction neuronal network during embryogenesis to mediate in- Published under the PNAS license. tercellular calcium signaling to induce AWC asymmetry (19, 20). 1Y.-W.H. and R.X. contributed equally to this work. In addition, NSY-5 gap junction protein and NSY-4 claudin-like 2To whom correspondence may be addressed. Email: [email protected]. protein function in parallel to suppress calcium signaling in the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ON AWC neuron via voltage- and calcium-activated SLO BK doi:10.1073/pnas.1908168116/-/DCSupplemental. potassium channels (19, 21, 22). It remains unknown what other First published November 25, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1908168116 PNAS | December 10, 2019 | vol. 116 | no. 50 | 25137–25146 Downloaded by guest on September 25, 2021 the GPCR gene srsx-3 (15, 16) (Fig. 1 A, i and B). Gain-of-function The vy10 lesion was identified as a G to A mutation, resulting mutations in the BK potassium channel gene slo-1 result in ex- in a glycine to arginine change, in the second exon of both pression of str-2p::TagRFP in 2 AWC neurons (2AWCON pheno- R06A4.4a/imb-2a and R06A4.4c/imb-2c isoforms (SI Appendix, type) (15). We identified the vy10 allele from a forward genetic Fig. S1B). imb-2 encodes an importin β-type nuclear transport screen for mutants that suppress the 2AWCON phenotype in receptor that mediates import of cargo into the nu- slo-1(ky399gf) mutants. The vy10 mutant lost expression of cleus (24). There are 3 predicted importin β in C. elegans str-2p::TagRFP and, instead, expressed srsx-3p::GFP in both (wormbase.org), 12 in Drosophila melanogaster (flybase.org), at AWC neurons (2AWCOFF phenotype) from the first larval stage (L1) least 10 in mice (Mouse Genome Informatics), and more than through adulthood (Fig. 1 A, ii and B). Since maintenance mutants 20 in humans (24, 25). C. elegans IMB-2 and human transportin display wild-type AWC asymmetry at the L1 stage but show a 1 are 52% identical and 87% similar throughout the entire pro- defect in AWC asymmetry later in development, these results teins (Fig. 2A and SI Appendix,Fig.S2). Human transportin 1 is a suggest that the vy10 mutation affects the initial establishment of superhelical protein, consisting of 20 HEAT repeats with adjacent AWC asymmetry. repeats connected by a linker (UniProt) (26–28). Similar to other The expression of 2 general AWC cell identity markers IMB-2 and transportin proteins, C. elegans IMB-2 protein has a odr-1p::TagRFP and ceh-36p:;TagRFP was not affected in vy10 predicted IBN_N -bonding domain and a HEAT-like repeat mutants (SI Appendix,Fig.S1A). These results suggest that the [Pfam (29)]. The predicted HEAT-like repeat of C. elegans IMB- vy10 mutation does not affect generalAWCidentity;ratherit 2 and human transportin 1 are 73% identical and 93% similar. The results in a defect in the induction of AWC asymmetry. glycine residue affected by the vy10 mutation is conserved in the vy10 mutants chemotaxed normally to the AWCOFF-sensed odor predicted HEAT-like repeat of IMB-2 and transportin proteins (SI 2,3-pentanedione but had a significant reduction in their ability to Appendix,Fig.S1C). This conserved glycine residue is located in chemotax toward the AWCON-sensed odor butanone (P < 0.001) the linker between HEAT repeats 9 and 10 of human transportin (Fig. 1C). Taken together, our results show that the vy10 mutation 1. imb-2 has 3 alternatively spliced isoforms (wormbase.org)(SI results in a loss of the AWCON cell identity at both molecular and Appendix,Fig.S1B). The predicted HEAT-like repeat is present in functional levels. IMB-2a and IMB-2c but is absent in IMB-2b. Both a PCR-amplified imb-2p::imb-2 genomic DNA frag- vy10 Is a Missense Mutation in imb-2/Transportin 1. We identified ment and an imb-2 fosmid clone almost completely rescued the the molecular lesion in vy10 mutants with 1-step whole-genome 2AWCOFF mutant phenotype in vy10 mutants (Fig. 1B and SI sequencing and single-nucleotide polymorphism mapping (23). Appendix,Fig.S1B). Together, these results support that the vy10

A str-2p::TagRFP; srsx-3p::GFP iidendrite i* AWCOFF iiiAWCON cilia * AWCOFF * axon * ON AWC AWCON AWCOFF * * Wild type imb-2(vy10) imb-2(OE)

B Marker expression in # of AWC C Marker Genetic background None One Both n i p < 0.001 Wild type L1 105 Wild type 108 1.0 imb-2(vy10) L1 111 0.8 imb-2(vy10) 161 imb-2(vy10)/+ 114 0.6 AWC imb-2 CRISPR-Cas9 132 vy10; imb-2p::imb-2 153 0.4 vy10; imb-2 fosmid 329 N O 0.2 C vy10; imb-2 fosmid::GFP 100 vy10; odr-3p::imb-2a 244 str-2p::GFPAW 0.0 vy10; odr-3p::imb-2b 181 Relative chemotaxis index Wild type imb-2(vy10) vy10; odr-3p::imb-2c 187 vy10; odr-3p::Transportin 1 110 odr-3p::imb-2a 117 bu (AWC ON -sensed odor) odr-3p::imb-2b 211 pd (AWC OFF -sensed odor) odr-3p::imb-2c 229 imb-2::mNG knock-in 105

Wild type OFF 109 C imb-2(vy10) 102 W srsx-3p::GFPA 020406080100%

Fig. 1. imb-2 is required and sufficient to promote the AWCON subtype. (A) Images of wild-type (i), imb-2(vy10) mutants (ii), and imb-2(OE)animals(iii)expressing the transgene str-2p::TagRFP (AWCON marker); srsx-3p::GFP (AWCOFF marker) in the adult stage. srsx-3p::GFP is also expressed in 2 AWB neurons. Asterisks indicate AWB cell bodies. (Scale bar, 10 μm.) (B)ExpressionofAWCON and AWCOFF markers in adults, unless otherwise indicated. n, total number of animals scored. (C) Quantification of the chemotaxis index. bu, butanone; pd, 2,3-pentanedione. Student’s t test was used to determine the P value. Error bars represent SEM.

25138 | www.pnas.org/cgi/doi/10.1073/pnas.1908168116 Alqadah et al. Downloaded by guest on September 25, 2021 52% identical, 87% similar A imb-2(vy10) G R HEAT IMB-2a IBN_N -like 883 aa Transportin 1 898 aa (Hs) Ran-binding domain 73% identical, 93% similar Heat repeats 43% identical, 89% similar

B Genetic background 2AWC OFF 1AWCON /1AWCOFF 2AWC ON n Wild type 115 imb-2(vy10) 161 slo-1(ky399gf) 111 slo-1(ky399gf); imb-2(vy10) 136 unc-36(e251) 124 unc-36(e251); imb-2(vy10) 220 tir-1(tm3036) 134 tir-1(tm3036); imb-2(vy10) 108 sek-1(ag1) 124 sek-1(ag1); imb-2(vy10) 119 020406080100% C nsy-5 slo-1 and slo-2 str-2 Innexin K + channel tir-1 unc-43 nsy-1 sek-1 imb-2 TIR-1/Sarm1 AWCOFF CaMKII MAPKKK MAPKK Transportin nsy-4 unc-2/unc-36 adaptor protein srsx-3 (default) Claudin egl-19/unc-36 Ca 2+ channel

nsy-5 slo-1 and slo-2 DEVELOPMENTAL BIOLOGY str-2 Innexin K + channel tir-1 unc-43 nsy-1 sek-1 imb-2 ON TIR-1/Sarm1 AWC CaMKII MAPKKK MAPKK Transportin (induced) nsy-4 unc-2/unc-36 adaptor protein srsx-3 Claudin egl-19/unc-36 Ca 2+ channel

Fig. 2. imb-2/transportin 1 acts downstream of the calcium-activated MAPK pathway in promoting AWCON.(A) Structure of C. elegans IMB-2a and human transportin 1 proteins. Hs, Homo sapiens.(B) Double mutant analysis of imb-2(vy10) with 2AWCON mutants. Animals were scored at the adult stage. n, total number of animals scored. (C) The AWC asymmetry genetic pathway that demonstrates imb-2/transportin 1 acting downstream of the calcium-activated MAPK pathway to promote AWCON. Genes in green represent AWCOFF promoting; genes in red represent AWCON promoting; and those in gray represent less active or inactive genes.

2AWCOFF mutant phenotype was caused by the identified mis- Appendix, Fig. S1B) caused early embryonic arrest. This prevented sense mutation in imb-2. us from scoring AWC phenotypes in the potential imb-2 null Both odr-3p::imb-2a and odr-3p::imb-2c,expressingimb-2 cDNA mutants. We also observed a high penetrance of embryonic lethal isoforms from an AWC odr-3 promoter (30) (SI Appendix,Fig. phenotype in RNA interference (RNAi) knockdown of imb-2, and S1B), rescued the vy10 2AWCOFF phenotype to a degree similar to we did not observe any defective AWC phenotypes in the survivors imb-2 genomic clones (Fig. 1B). However, odr-3p::imb-2b only of imb-2(RNAi) animals. Unlike imb-2 deletion mutants and se- partially rescued the vy10 2AWCOFF phenotype. These results vere imb-2(RNAi) animals, imb-2(vy10) mutants are viable and suggest that imb-2 mainly acts in AWC cells to regulate AWC fertile, suggesting that imb-2(vy10) is likely not a null allele. To asymmetry and suggest an important role of the HEAT-like repeat circumvent the embryonic lethal phenotypes of imb-2 null alleles, of IMB-2 in AWC asymmetry. In addition, the transgene expressing we generated conditional imb-2 knockdown or knockout inser- human transportin 1 cDNA from the AWC odr-3 promoter res- tion and deletion mutations in AWC neurons using CRISPR-Cas9 cued the vy10 mutant asymmetry phenotype, similar to the rescuing technology (31). Transgenic animals of AWC imb-2 CRISPR-Cas9, ability of odr-3p::imb-2a and imb-2c (Fig. 1B and SI Appendix, Fig. expressing Cas9 under the AWC odr-3 promoter and an imb-2 S1B). This result suggests conservation of imb-2/transportin 1 gene single guide RNA (target site in exon 2 of imb-2a and imb-2c) function in C. elegans and humans. Furthermore, overexpression of under a U6 small nuclear RNA promoter, recapitulated the odr-3p::imb-2a, odr-3p::imb-2b,andodr-3p::imb-2c in wild-type 2AWCOFF phenotype observed in imb-2(vy10) mutants (Fig. 1B animals resulted in a high penetrance of the 2AWCON pheno- and SI Appendix,Fig.S1B). Together, these results further support type, opposite to the 2AWCOFF phenotype of the vy10 mutants that the vy10 mutation is a partial loss-of-function allele of imb-2 in (Fig. 1 A, iii and B). In addition to the 2AWCON phenotype, AWC asymmetry. overexpression of odr-3p::imb-2c also caused a high penetrance of the 2AWCOFF phenotype (Fig. 1B). Taken together, these results imb-2 Acts Downstream of a Calcium-Activated MAP Kinase Cascade suggest that imb-2 is essential and sufficient to promote the to Promote AWCON. imb-2(vy10) was identified as a suppressor of AWCON cell identity and that the vy10 mutation causes loss of or slo-1(ky399gf) mutants, suggesting imb-2 acts downstream of slo-1 reduction of imb-2 function in AWC asymmetry. Heterozygous to promote AWCON (Fig. 2 B and C). The position of imb-2/ imb-2(vy10/+) animals displayed wild-type AWC asymmetry (Fig. transportin 1 in the AWC asymmetry genetic pathway was further 1B), indicating that the imb-2(vy10) allele is recessive. determined by double mutant analysis of imb-2(vy10) with 2AWCON Both imb-2(tm6328) and imb-2(tm6405) alleles in which partial mutants. The 2AWCON phenotype of unc-36(e251) (calcium chan- exon and intron regions of all 3 imb-2 isoforms are deleted (SI nel subunit), tir-1(tm3036) (Sarm 1 adaptor protein), and sek-1(ag1)

Alqadah et al. PNAS | December 10, 2019 | vol. 116 | no. 50 | 25139 Downloaded by guest on September 25, 2021 A imb-2::mNG knock-in; odr-1p::TagRFP

Three-fold stage embryo First-stage larva Adult

B imb-2::mNG knock-in C 100 imb-2::mNG knock-in odr-1p::TagRFP ns (p = 0.99) 80 p < 0.001 ns (p = 0.63) ns (p = 0.3) 60 p = 0.04

AWCL>AWCR 40 of animals AWCL=AWCR % 20 AWCL

0 0 0

Wild type n=20 n=31 nsy-5(ky634)nsy-4(ky627) n=20 n=20slo-1(lf);slo-2(lf)slo-1(ky399gf)n=24 unc-43(n498gf)n=20 D imb-2::mNG knock-in E str-2p::TagRFP p = 0.006 imb-2::mNG knock-in ceh-36p::myrTagRFP 100 p < 0.001

80

n=26 60

of animals 40 %

20

0 0 AWC ON > AWCOFF AWC ON = AWCOFF AWC ON < AWCOFF

imb-2(vy10); imb-2 fosmid(OE) F 100 G 100 80 n=147 2AWC ON 80 p < 0.0001

60 1AWC OFF / 60 1AWCON 40 2AWC OFF 40 % of animals

20 % of animals 20 0 0 imb-2(vy10) cell AWC OFF AWC OFF AWC ON AWC ON Wild type b-2(vy10) (n=108) OFF ON OFF ON im (n=305)imb-2(vy10); imb-2(OE) cell AWC AWC AWC AWC (n=329) imb-2 fosmid(OE) Mosaic animals: One AWC cell is imb-2(vy10) the other AWC cell is imb-2(OE)

Fig. 3. imb-2/transportin 1 acts cell autonomously to promote the AWCON subtype. (A)Imagesofimb-2::mNG knock-in expression in a 3-fold stage embryo (i), a first-stage larva (ii), and an adult (iii). AWC neurons were labeled with odr-1p::TagRFP. AWC cell bodies are outlined with dashed lines. (Scale bars, 5 μm[i and ii]and50μm[iii].) Asterisks indicate nonadult animals. (B)Imagesofimb-2::mNG knock-in expression at a higher level in the AWCL neuron than in AWCR at the L1 stage (ventral view). Both AWCL and AWCR were marked by odr-1p::TagRFP. (Scale bar, 5 μm.) (C) Quantification of asymmetric imb-2::mNG knock-in expression in AWCL and AWCR neurons. No significant difference was observed between AWCL > AWCR and AWCL < AWCR in wild-type animals. ns, not significant. n, total number of animals scored. P values were calculated using Fisher’s exact test. Error bars represent SE of proportion. (D)Imagesofimb-2::mNG knock-in expression at a higher level in AWCON than in AWCOFF inaL1animal(dorsalview).AWCON was marked by str-2p::TagRFP and ceh-36p::myrTagRFP,whileAWCOFF was only marked by ceh-36p::myrTagRFP. (Scale bar, 5 μm.) (E) Quantification of asymmetric expression of imb-2::mNG knock-in in AWCON and AWCOFF. n, total number of animals scored. P values were determined using a Z test. Error bars represent SE of proportion. (F) Quantification of AWC asymmetry phenotypes in wild-type, imb-2(vy10),andimb-2(vy10) mutants containing the extrachromosomal transgene imb-2 fosmid(OE); odr-1p::DsRed.(G) Quantification of AWC phenotypes in imb-2(vy10) mosaic animals containing the extrachromosomal transgene imb-2 fosmid(OE) in only 1 AWC neuron, inferred by the presence of the coinjected odr-1p::DsRed AWC marker. The data were obtained from a subset of animals scored in F.

25140 | www.pnas.org/cgi/doi/10.1073/pnas.1908168116 Alqadah et al. Downloaded by guest on September 25, 2021 (MAPKK) mutants was suppressed by the 2AWCOFF phenotype ogous recombination (31–33) (SI Appendix,Fig.S1B). imb- of imb-2(vy10) mutants. These results suggest that imb-2/transportin 2::mNG knock-in animals displayed wild-type AWC asym- 1 acts downstream of the MAP kinase cascade to promote the metry (Fig. 1B), indicating that the tagged IMB-2::mNG AWCON subtype (Fig. 2C). fusion protein is functional. IMB-2::mNG was detected in the nucleus and around the nuclear envelope of numerous imb-2/Transportin 1 Is Asymmetrically Expressed in AWCON Neurons. cells in the head and body during embryogenesis, larval stages, The expression pattern of imb-2 was analyzed in imb-2::mNG and adulthood (Fig. 3A). The broad expression pattern of knock-in (where [mNG] represents mNeonGreen) animals in imb-2 in the head is consistent with expression of vertebrate which we tagged the C-terminal end of endogenous IMB-2 with transportin 1 in multiple regions of the brain, including the ol- the fluorescent reporter mNG using Cas9-triggered homol- factory bulb (34).

A odr-3p::nsy-7::GFP; odr-1p::TagRFP GFP Merge AWC cell body odr-3p::nsy-7::GFP

2.7 kb odr-3 promoter unc-54 3’ UTR Wild type nucleus nsy-7 GFP

1 kb

imb-2(vy10)

odr-3p::nsy-7::2xnlsGFP; odr-1p::TagRFP GFP Merge odr-3p::nsy-7::2xnlsGFP DEVELOPMENTAL BIOLOGY 2.7 kb odr-3 promoter unc-54 3’ UTR imb-2(vy10) nsy-7 2xnlsGFP

B str-2p::GFP expression Genetic background 2AWCOFF 1AWCON /1AWCOFF 2AWCON n Wild type 108 nsy-7(tm3080) L1 100 nsy-7(tm3080) 100 odr-3p::nsy-7 155 imb-2(vy10) 161 vy10; odr-3p::nsy-7::GFP 121 vy10; odr-3p::nsy-7::2xnlsGFP 112 020406080100% C Venus Venus odr-1p::2xnlsTagRFP odr-3p::imb-2a::VN173

2.7 kb odr-3 promoter IMB-2 unc-54 3’ UTR imb-2a cDNA VN + NSY-7 odr-3p::imb-2a(vy10)::VN173 vy10 (G417R) 2.7 kb odr-3 promoter unc-54 3’ UTR imb-2a cDNA VN IMB-2G417R

+ NSY-7 odr-3p::nsy-7::VC155 2.7 kb odr-3 promoter unc-54 3’ UTR nsy-7 VC

IMB-2 odr-3p::nsy-7(ky630)::VC155 H179Y ky630 (H179Y) + NSY-7 2.7 kb odr-3 promoter unc-54 3’ UTR nsy-7 VC 1 kb

Fig. 4. imb-2 is required for nuclear localization of NSY-7 homeodomain-like transcription factor in the specification of AWCON identity. (A) Images of NSY- 7::GFP and NSY-7::2xnlsGFP expressed from single copy insertion transgenes odr-3p::nsy-7::GFP and odr-3p::nsy-7::2xnlsGFP, respectively, in AWC at the L1 stage. AWC neurons were labeled with odr-1p::TagRFP. (Scale bar, 5 μm.) (B) Quantification of AWC asymmetry phenotypes in L1 or adults. (C) Images of transgenic animals for bimolecular fluorescence complementation (BiFC) assays between different forms of IMB-2 and NSY-7 proteins, each fused to non- fluorescent fragments of Venus, at the L1 stage. (Scale bar, 5 μm.) VN, VN173 (Venus 1–172); VC, VC155 (Venus 155–238).

Alqadah et al. PNAS | December 10, 2019 | vol. 116 | no. 50 | 25141 Downloaded by guest on September 25, 2021 IMB-2::mNG was detected in both AWC neurons in late SV40) on the same locus of a . NSY-7::2xnlsGFP embryogenesis during which AWC asymmetry was established was localized to the AWC nucleus in imb-2(vy10) mutants (Fig. and was maintained until adulthood (Fig. 3A). IMB-2::mNG was 4A), suggesting that the SV40 NLS bypassed the requirement asymmetrically expressed in the left AWC neuron (AWCL) or of IMB-2 for the transport of NSY-7::2xnlsGFP to the nucleus. the right AWC neuron (AWCR) in a stochastic manner (Fig. 3 B The imb-2(vy10) 2AWCOFF mutant phenotype was not rescued and C). Stochastic asymmetry of imb-2 expression in AWC neurons by cytoplasmic localized NSY-7::GFP. However, nuclear local- is consistent with the random nature of AWC asymmetry. The ized NSY-7::2xnlsGFP not only rescued the imb-2(vy10) percentage of animals with equivalent imb-2 expression in AWCL 2AWCOFF mutant phenotype, but also resulted in a 2AWCON and AWCR was significantly higher in nsy-5(ky634) (gap junction) phenotype. These results suggest that imb-2 is required for the and slo-1(ky399gf) (BK potassium channel) mutants (P < 0.001 and transport of NSY-7 into the nucleus of AWC neurons to induce P = 0.04, respectively) (Fig. 3C). However, asymmetric imb-2 the AWCON cell identity. expression was not significantly affected in nsy-4(ky627) (claudin), slo-1(eg142lf);slo-2(ok2214lf) (BK potassium channels), or IMB-2/Transportin 1 Is in Close Proximity to NSY-7 in AWC Neurons. unc-43(n498gf) (CaMKII) mutants. These results suggest that To determine whether IMB-2/transportin 1 interacts with NSY-7 NSY-5 gap junctions and SLO-1 BK potassium channels regulate in AWC neurons, we used a bimolecular fluorescence comple- asymmetric expression of imb-2 in AWC neurons. mentation (BiFC) assay (39). Two nonfluorescent Venus frag- IMB-2::mNG was expressed at a significantly higher expres- ments, VN173 and VC155, were fused to IMB-2 and NSY-7, sion level in the AWCON neuron than the AWCOFF neuron respectively, and the odr-3 promoter was used to drive their ex- in the majority of animals (Fig. 3 D and E). Asymmetric expression pression in AWC neurons (Fig. 4C). Coexpression of odr-3p::imb- of imb-2 in AWCON wasalsoobservedinourinitialassessmentof 2::VN173 and odr-3p::nsy-7::VC155 transgenes resulted in punctate the imb-2 expression pattern using imb-2 fosmid::GFP in which we Venus fluorescence in the nucleus of AWC neurons (Fig. 4C and fused GFP with the C terminus of IMB-2 by homologous re- SI Appendix,Fig.S5A). Similarly, coexpression of odr-3p::imb- combination (35), that rescued imb-2(vy10) mutants (Fig. 1B and 2::VC155 and odr-3p::nsy-7::VN173 transgenes in which VC155 and SI Appendix, Figs. S1B and S3 A–C). These results are consistent VN173 were fused to IMB-2 and NSY-7, respectively, also showed with the hypothesis that imb-2 acts cell autonomously to promote a punctate pattern of Venus fluorescence in the AWC nucleus (SI the AWCON cell identity. Appendix,Fig.S5B). These BiFC results suggest that IMB-2 and NSY-7 proteins may be close enough to interact. The interaction imb-2/Transportin 1 Acts Cell Autonomously in AWCON Neurons. of IMB-2 with MLS-2 was tested as a negative control of this assay Expression of imb-2 fosmid(OE), odr-1p::DsRed or odr-3p::imb- since the nuclear localization of MLS-2 was not affected in imb- 2a(OE), odr-1p::DsRed transgenes in both AWC cells significantly 2(vy10) mutants (SI Appendix,Fig.S4B). As expected, we did rescued the 2AWCOFF phenotype of imb-2(vy10) mutants (Fig. 3F not observe Venus expression from the control assays (SI Ap- and SI Appendix,Fig.S3D). Spontaneous loss of the extrachro- pendix,Fig.S5C). mosomal array resulted in mosaic animals in which only 1 of the The nsy-7(ky630) allele is a missense mutation leading to ex- AWC neurons retained the imb-2(OE) rescuing transgene (inferred clusively cytoplasmic localization of the mutant NSY-7 protein by the AWC marker odr-1p::DsRed) and the other AWC cell (14) (SI Appendix, Fig. S5D). Venus expression was observed remained imb-2(vy10). In the majority of the mosaic animals that between IMB-2G417R with the vy10 mutation and NSY-7; however, exhibited rescue of the mutant phenotype (1 AWCON/1 AWCOFF), the localization pattern was mostly excluded from the nucleus (Fig. the AWC cell expressing imb-2 fosmid or odr-3p::imb-2a became 4 C, Middle). Similarly, Venus expression of IMB-2 and NSY- AWCON,andtheimb-2(vy10) AWC cell became AWCOFF (Fig. 3G 7H179Y with the ky630 mutation also resulted in localization out- and SI Appendix,Fig.S3E). These results suggest that imb-2 side of the AWC nucleus (Fig. 4 C, Bottom). These results suggest acts largely cell autonomously to induce AWCON. that imb-2(vy10) and nsy-7(ky630) mutations do not abolish the Mosaic analysis was also performed in transgenic lines in which interaction between IMB-2 and NSY-7 but rather prevent the odr-3p::imb-2a was overexpressed in a wild-type background, protein complex from entering the nucleus. resulting in a 2AWCON phenotype (SI Appendix,Fig.S3F). Importin α proteins function as adaptor proteins in the classical When the odr-3p::imb-2a array was present in only a single nuclear transport process by binding to importin β and the NLS of AWC neuron, the imb-2(OE) cell became AWCON,andthe cargoes (40, 41). In nonclassical nuclear import pathways, importin wild-type cell became AWCOFF in the majority of mosaic ani- β proteins can directly bind certain cargoes, such as transcription mals (SI Appendix,Fig.S3G). These data further support the factors and ribosomal proteins, independent of importin α (42). notion that imb-2 specifies the AWCON fate in a largely cell There are 3 importin α genes, ima-1, ima-2,andima-3 in the C. autonomous fashion. elegans genome (wormbase.org). All of ima-1, ima-2, and ima-3 deletion mutants analyzed displayed wild-type AWC asymmetry imb-2/Transportin 1 Is Required for Nuclear Localization of NSY-7 (SI Appendix,Fig.S6), suggesting that these importin α genes are Homeodomain-like Transcription Factor to Specify the AWCON not required for AWC asymmetry. Consistent with these results, Subtype. To identify the cargo of IMB-2/transportin in promoting RNAi knockdown of ima-1, ima-2,andima-3 individually or to- AWCON, we examined fluorescent reporters translationally fused gether did not cause abnormal AWC asymmetry phenotypes. with 5 candidate transcription factors that are implicated in AWC Together with the BiFC assay result that implies directly binding of asymmetry (14, 36–38) (SI Appendix,Fig.S4A). Nuclear localiza- IMB-2 to its cargo NSY-7, these results suggest that IMB-2 may tion of SOX-2, CEH-36 (OTX/OTD), MLS-2 (HMX/NKX), and mediate a nonclassical nuclear import of NSY-7 independent of DIE-1 (zinc finger) was not affected in imb-2(vy10) mutants (SI importin α to establish AWC asymmetry. Appendix,Fig.S4B). However, NSY-7 was detected in the cytosol and largely excluded from the nucleus of AWC neurons in imb- nsy-7 Is Required for sox-2 Expression to Promote the AWCON Subtype. 2(vy10) mutants (Fig. 4A). These results suggest that imb-2 is re- Previous studies have shown that nsy-7 functions to maintain AWC quired for the transport of NSY-7 into the nucleus of AWC neurons. asymmetry by studying nsy-7(ky630) mutants (14). We found that To directly determine the requirement of IMB-2 for nuclear nsy-7(tm3080) deletion mutants displayed a complete penetrance localization of NSY-7 in promoting the AWCON subtype, we of the 2AWCOFF phenotype at both L1 and L4 stages (Fig. 4B and generated Mos1-mediated single copy insertion transgenes of SI Appendix,Fig.S5D), revealing an additional role of nsy-7 in odr-3p::nsy-7::GFP or odr-3p::nsy-7::2xnlsGFP (containing the establishment of AWC asymmetry besides maintenance. 2 copies of the nuclear localization signal [NLS] derived from Overexpression of nsy-7 in AWC caused a strong 2AWCON

25142 | www.pnas.org/cgi/doi/10.1073/pnas.1908168116 Alqadah et al. Downloaded by guest on September 25, 2021 A sox-2ps::2xnlsGFP; odr-1p::TagRFP B GFP Merge Genetic sox-2ps::2xnlsGFP expression background 0AWC 1AWC 2AWC n

Wild type Wild type L1 106

nsy-7(tm3080) L1 143

imb-2(vy10) L1 104

nsy-7(tm3080)

Wild type L4 104

nsy-7(tm3080) L4 133

imb-2(vy10) L4 126 imb-2(vy10) 020406080100%

C str-2p::GFP expression D Expression of marker Row Genetic background 0AWC 1AWC 2AWC n Marker 012AWCn

a Wild type L1 105 sox-2ps::2xnlsGFP 118 b sox-2 fosmid::mCherry L1 104 sox-2ps(NSY-7m):: *** c imb-2(vy10) L1 111 62 2xnlsGFP

d imb-2(vy10); sox-2 fosmid::mCherry L1 108 020406080100% DEVELOPMENTAL BIOLOGY e nsy-7(tm3080) L1 100 sox-2ps::2xnlsGFP f nsy-7(tm3080); sox-2 fosmid::mCherry L1 106 NSY-7 site unc-54 3’ UTR 2xnlsGFP g Wild type L4 108 2.8 kb upstream h sox-2 fosmid::mCherry L4 117 sox-2ps (NSY-7m)::2xnlsGFP i imb-2(vy10) L4 161 mutated NSY-7 site unc-54 3’ UTR j imb-2(vy10); sox-2 fosmid::mCherry L4 125 X 2xnlsGFP k nsy-7(tm3080) L4 100 2.8 kb upstream l nsy-7(tm3080); sox-2 fosmid::mCherry L4 127 1 kb CCTTAAC X C A GCTG G 020406080100%

E Unlabeled sox-2p-wt Unlabeled sox-2p-m F 6xHis-NSY-7 - + + + + +++++ 1.3 IRDye-sox-2 p-wt + + +++ + +++ + Lanes 12345 678910

t 1.1

NSY-7-DNA i

n

U yr

a 0.9

r

tib r A 0.7 DNA 0.5 NSY-7 site 020406080 sox-2p-wt GCTAACCATCAAACCT CCTTAAC TCAATTGCTCGTGCAA Fold competitor sox-2p-m GCTAACCATCAAACCT C A GCTG G TCAATTGCTCGTGCAA wild type mutant C. remanei TCTAACCATCGAACCT CCTTAAC TCAATTGCTCGTGCAA C. briggsae TCAAAGCATCGAACCT CCTTAAC TCAATTGCTCGTGCAA C. brenneri TTTAACCATCGAACCT CCTTAAC TCAATTGCTCGTGCAA

Fig. 5. nsy-7 is required for sox-2 expression in promoting the AWCON subtype. (A) Images of sox-2ps::2xnlsGFP expression at the L1 stage. (Scale bar, 5 μm.) Asterisks indicate AWB cell body. (B) Quantification of the percentage of animals expressing sox-2ps::2xnlsGFP in AWC at the L1 and L4 stages. (C) Quan- tification of AWC asymmetry phenotypes at the L1 and L4 stages. (D) Quantification of sox-2ps::2xnlsGFP and sox-2ps(NSY-7m)::2xnlsGFP expression in AWC at the L1 stage. NSY-7m, mutated NSY-7-binding site within the sox-2 upstream regulatory sequence. ***P < 0.0001. Statistic comparison was performed by Fisher’s exact test. Quantification of the transgene expression in each of the independent lines is included in SI Appendix, Fig. S7.(E) A representative gel image of electrophoretic mobility shift assays (EMSA) with 6xHis-tagged NSY-7 protein and an IRDye-labeled DNA probe containing the NSY-7 consensus- binding site in the sox-2 promoter. Unlabeled wild-type or mutant competitor probes were added to lanes 3–6 and 7–10, respectively at increasing con- centrations (20×,40×,60×, and 80×). Competitive-binding assays were performed 9 independent times, and these independent assays showed the same trend that the mutant competitor probe was not as efficient at competing away the NSY-7-DNA complex as the wild-type competitor probe. Nucleotides in gray are sequences of Caenorhabditis remani, Caenorhabditis briggsae, and Caenorhabditis brenneri that differ from the C. elegans sequence. Sequence alignment between species was performed on http://genome.ucsc.edu.(F) Relative band intensities of the NSY-7-DNA complex were plotted against the concentration of the competitor probe. Images and respective band intensity plots from 2 other independent EMSA assays are presented in SI Appendix, Fig. S8.

Alqadah et al. PNAS | December 10, 2019 | vol. 116 | no. 50 | 25143 Downloaded by guest on September 25, 2021 phenotype (Fig. 4B), suggesting that nsy-7 is sufficient to induce A Wild type the AWCON identity. The HMG-box transcription factor sox-2 is a candidate target IMB-2 of NSY-7 since sox-2 has been implicated in promoting the IMB-2 NSY-7 AWCON identity (37) and contains a potential NSY-7 binding site, previously identified by protein-binding microarrays (14), in the upstream regulatory sequence. An integrated sox-2 transcrip- NSY-7 IMB-2 tional reporter transgene, sox-2ps::2xnlsGFP (37), was expressed in IMB-2 both AWC neurons at the L1 stage in the majority of wild-type animals (Fig. 5 A and B). The number of AWC neurons expressing NSY-7 sox-2ps::2xnlsGFP was significantly reduced in nsy-7(tm3080) and imb-2(vy10) mutants, indicating that nsy-7 and imb-2 are required NSY-7 bs sox-2 NSY-7 bs sox-2 for sox-2 expression in AWC at the L1 stage. However, sox-2 ex- Nucleus Nucleus pression was lost in both AWC neurons of wild-type, nsy- 7(tm3080),andimb-2(vy10) mutants at the L4 stage (Fig. 5B). AWCON AWCOFF Together, these results suggest that IMB-2 transports NSY-7 into the nucleus of AWC neurons to activate sox-2 expression at the B imb-2(vy10) L1 stage but not at the L4 stage. Overexpression of sox-2 fosmid::mCherry almost completely IMB-2 OFF IMB-2 NSY-7 G417R rescued the 2AWC phenotype of imb-2(vy10) and nsy-7(tm3080) G417R mutants at the L1 stage (Fig. 5C, row d compared with row c and X row f compared with row e, respectively). However, the rescue ability of sox-2 fosmid::mCherry was greatly reduced in imb-2(vy10) mutants and completely lost in nsy-7(tm3080) mutants at the L4 stage (Fig. 5C, rows i and j and rows k and l). These results are consistent with sox-2 expression in AWC at L1 but not in L4 (Fig. 5B). The data also suggest that nsy-7 activates sox-2 expression for NSY-7 bs sox-2 sox-2 the establishment of AWC asymmetry during early development NSY-7 bs and regulates other factors for the maintenance of AWC asym- Nucleus Nucleus metry later in development. NSY-7 was previously described as a OFF repressor of the AWC -specific marker srsx-3 expression to AWCOFF AWCOFF maintain the AWCON subtype (14). Our results reveal a role of NSY-7 as a transcriptional activator of sox-2 expression in the Fig. 6. Model of imb-2 function in AWC asymmetry. (A) In wild-type animals, specification of the AWCON identity. imb-2 and nsy-7 are asymmetrically expressed in the AWC neuron that becomes AWCON. IMB-2 binds to NSY-7 and mediates the transport of NSY-7 into the ON NSY-7 Transcription Factor Binds to sox-2 Upstream Regulatory nucleus to activate sox-2 expression thereby inducing the AWC identity. (B)In imb-2(vy10) mutants, NSY-7 fails to enter the nucleus, leading to loss of sox-2 Sequence. The NSY-7 target site CCTTAAC, identified by protein- OFF binding microarrays (14), is located in the upstream regulatory expression and a 2AWC phenotype. Gray, less active or inactive molecules or expression. sequence of sox-2 (Fig. 5 D and E). The extrachromosomal trans- gene sox-2ps::2xnlsGFP, containing the putative NSY-7-binding site, was expressed in, at least, 1 AWC neuron in the majority of findings (14), only 1 of the AWC neurons expressed nsy-7p::GFP animals (Fig. 5D and SI Appendix,Fig.S7, 10 lines examined). The in those transgenic animals that displayed GFP expression in number of AWC neurons expressing sox-2ps(NSY-7m)::2xnlsGFP, AWC cells (SI Appendix,Fig.S9B). Asymmetric expression of nsy- containing a mutated NSY-7-binding site, was significantly de- 7p::GFP in AWCL or AWCR was stochastic (SI Appendix, Fig. creased (Fig. 5D and SI Appendix,Fig.S7, 6 lines examined) S9C). Neither stochastic asymmetry of nsy-7 expression in AWC compared to that of AWC expressing sox-2ps::2xnlsGFP.These neurons or the number of AWC expressing nsy-7p::GFP was af- results suggest that the NSY-7-binding site is required for appro- fected in imb-2(vy10) mutants (SI Appendix,Fig.S9C and D). nsy-7 priate expression of sox-2 in AWC neurons. was exclusively expressed in AWCON neurons, while no expres- In EMSA, 6xHis-tagged NSY-7 was able to bind an IRDye- sion was observed in AWCOFF cells (SI Appendix,Fig.S9E and F). labeled DNA probe containing 39 bp of the sox-2 upstream reg- This result further suggests that the NSY-7 transcription factor ulatory sequence, which includes the NSY-7 consensus site (Fig. ON 5E and SI Appendix,Fig.S8A and C, lane 2). A wild-type un- promotes the AWC identity in a cell-autonomous manner. labeled probe (sox-2p-wt) was able to compete away NSY-7 from The expression pattern of sox-2 was examined in sox-2::mNG the IRDye-labeled probe, whereas the mutated unlabeled probe knock-in animals in which we tagged the C-terminal end of en- dogenous SOX-2 with mNG using Cas9-triggered homologous re- (sox-2p-m), containing the same mutated NSY-7 consensus site as – in sox-2ps(NSY-7m)::2xnlsGFP,wasnotasefficientasacompetitor combination (31 33) (SI Appendix,Fig.S9A). Like imb-2 and nsy-7, – – sox-2 was asymmetrically expressed at a higher level in AWCON (Fig. 5 E and F and SI Appendix,Fig.S8A D,lanes3 6 compared OFF to lanes 7–10). These results suggest that NSY-7 binding was neurons than in AWC neurons in a stochastic manner (SI Ap- – sox-2 pendix,Fig.S9G J). Together, these results support the role of nsy- specific to the upstream regulatory sequence of . Together ON with the requirement of nsy-7 for expression of sox-2, these results 7 in activating sox-2 expression to promote the AWC identity. further support the role of NSY-7 as a transcriptional activator, Discussion whereas it was previously reported as a repressor (14). Here, we identify an essential role of imb-2/transportin 1 in a sto- nsy-7 and sox-2 Are Asymmetrically Expressed in the AWCON Neuron. chastic choice of asymmetric olfactory neuron subtypes in C. elegans The expression pattern of nsy-7 in AWC neurons was further from an unbiased forward genetic screen. We show that imb-2/ analyzed in transgenic animals expressing a nsy-7p::GFP extra- transportin 1 mediates transport of the NSY-7 homeodomain-like chromosomal transgene in which GFP was driven by a 21 kb nsy-7 transcription factor into the nucleus of AWC olfactory neurons, promoter (14) (SI Appendix,Fig.S9A). Consistent with previous which, in turn, activates sox-2 expression to promote the AWCON

25144 | www.pnas.org/cgi/doi/10.1073/pnas.1908168116 Alqadah et al. Downloaded by guest on September 25, 2021 subtype. This study implicates karyopherins in the establishment of functions. It is possible that the glycine to arginine change within stochastic cell identity choice and left–right patterning. As C. the HEAT-like repeat in vy10 mutants specifically affects nuclear elegans imb-2 is highly conserved with human transportin 1, this import of the NSY-7 homeodomain-like transcription factor for process may prove to be conserved in establishing stochastic cell a subset of imb-2 functions including establishing stochastic identity and left–right asymmetry in mammals. choice of AWC subtypes. Our results suggest a mechanistic model for imb-2 function in Although karyopherins have been implicated in various cell a stochastic choice of AWC olfactory neuron subtypes (Fig. 6). In biological and developmental processes, the majority of implica- wild type, imb-2, nsy-7, and sox-2 are asymmetrically expressed tions derive from RNAi knockdown of karyopherins, expression of in AWCON in a stochastic manner (Fig. 6A). IMB-2 binds to karyopherins during particular cell biological events or in partic- NSY-7 and mediates nuclear transport of NSY-7 in AWCON ular tissues, or mutational analyses of candidate cargo proteins. neurons. Nuclear-localized NSY-7 directly activates expression For example, RNAi knockdown of imb-2 prevents redox- of sox-2 to promote the AWCON identity. In imb-2(vy10) mu- dependent nuclear import of the transcription factor FOXO/ tants, as in wild type, nsy-7 is asymmetrically expressed in the DAF-16 (47) and suppresses the life span of long-lived mito- AWC neurons in a stochastic manner (Fig. 6B). However, NSY- chondrial mutants with increased expression of FOXO/DAF- 7 is largely excluded from the nucleus of AWC neurons in imb- 16 target genes (48). Defective nuclear transport of key cellular G417R 2(vy10) mutants. Although IMB-2 (containing the vy10 regulator molecules has been reported in a variety of diseases mutation) and NSY-7 are still in close proximity, the imb-2(vy10) and cancers; however, the majority of pathogenic mutations are mutation abolishes the ability of the IMB-2-NSY-7 complex to identified in the cargo proteins rather than the karyopherins enter the nucleus. Thus, sox-2 expression is subsequently lost, that transport them (49–51). Although deregulation and/or ON resulting in the inability to promote the AWC identity and a mutations of karyopherins have been reported in some cancers, OFF 2AWC mutant phenotype in imb-2(vy10) mutants. the molecular mechanisms of these karyopherin mutations and It was previously shown that NSY-7 responds to transient how the mutations lead to cancers remain to be elucidated embryonic signaling of the NSY-5 gap junction neuronal network (49). Our study sets a precedent by mechanistically implicating by acting as a transcriptional repressor (repressing AWCOFF ON a widely expressed karyopherin protein in a specific function of genes in AWC neurons) in the maintenance of AWC asym- a multicellular organism directly through the analysis of mis- metry (14). Our results reveal a role of NSY-7 as a transcrip- sense karyopherin mutants identified from an unbiased forward tional activator of sox-2 expression in establishing the stochastic ON genetic screen. Our findings reveal structure-function insight choice of the AWC subtype. Our study extends the previous into a conserved amino acid residue of karyopherins in a sto- model of AWC asymmetry by identifying a role of imb-2/trans- chastic choice of cell identity and left–right patterning. DEVELOPMENTAL BIOLOGY portin 1 in the coupling transient NSY-5 gap junction signaling from the cell membrane and the cytosol to the NSY-7-SOX-2 Materials and Methods transcriptional cascade in the nucleus. Strains and Transgenes. The wild-type C. elegans strain is N2, Bristol variety. In nuclear import pathways, importin β interacts with the nu- Strains were maintained by standard methods (52). A list of strains and clear pore complex to promote selective and efficient transport of transgenes is included in SI Appendix, Supplemental Materials and Methods. importin-cargo complexes across the nuclear envelope into the nucleus (43, 44). Once importin-cargo complexes enter the nu- Isolation of imb-2(vy10) Mutants. A forward genetic screen was performed as cleus, binding of GTP-bound Ran GTPase to importin β induces previously described (52). kyIs140I; slo-1(ky399gf) P0 mutants were treated structural changes in importin β, leading to the release of cargoes with EMS. Ten F1 progenies were picked onto single plates, and F2 were ON (24). Our study suggests that the conserved glycine in the HEAT- screened for suppression of the slo-1(ky399gf) 2AWC phenotype using a like repeat mutated in the vy10 mutant may not be required for Zeiss fluorescence dissecting microscope. The vy10 mutation was identified IMB-2 to recognize and bind to the cargo NSY-7 in the cytoplasm from a screen of 5,700 genomes. but is important for transport of the IMB-2-NSY-7 complex across Whole-Genome Sequencing. The 1-step whole-genome-sequencing and SNP the nuclear envelope into the nucleus. It is possible that the gly- mapping strategy (23) was used to identify the vy10 mutation with an Illu- cine to arginine change in the vy10 mutants may affect the binding mina GAIIX sequencing platform and 100-nucleotide reads. Analysis of se- affinity of IMB-2 to the complex and, thus, abolishes quencing results was performed using CloudMap software as previously the ability of IMB-2-NSY-7 to enter into the AWC nucleus. described (53). In contrast with embryonic lethality of imb-2 deletion mutants and RNAi knockdown animals, imb-2(vy10) mutants are viable BiFC Assays. BiFC assays were performed as previously described (39). Non- and fertile. Our approach revealed a role of imb-2 in postmitotic fluorescent VN173 and VC155 fragments of Venus were fused to IMB-2 and diversification of olfactory neuron subtypes, which would have NSY-7, respectively. The fusion protein constructs, driven by the AWC odr-3 been masked by candidate gene approaches of analyzing the promoter, were coinjected into animals. Expression of Venus was imaged at phenotypes in imb-2 deletion mutants or imb-2 RNAi knock- the L1 stage. down animals. In humans, importin β proteins have been shown ACKNOWLEDGMENTS. We thank Cori Bargmann, Alex Boyanov, Oliver to carry a diverse set of cargoes into the nucleus. Crystal struc- Hobert, Dan Dickinson, Bob Goldstein, Jim Wells, David Crowe, Shohei tures of human importin β-1 bound with cargoes show that car- Mitani, WormBase, and C. elegans Genetic Center (funded by the NIH goes bind at different sites on importin β (45, 46). We propose Office of Research Infrastructure Programs P40 OD010440) for assistance, that broadly expressed IMB-2 mediate nuclear transport of se- strains, reagents, and/or protocols. This work was supported by the Na- tional Science Foundation Grant (IOS-1455758 to C.C.), a Whitehall Foun- lective cargoes for different functions and that functional speci- dation Research Award (to C.-F.C.), an Alfred P. Sloan Research Fellowship ficity of IMB-2 is conferred by different amino acid residues, (to C.-F.C.), and the National Institutes of Health Grants (5R01GM098026- which mediate nuclear transport of specific cargoes for distinct 05 to C.-F.C., R01GM111320 to C.C.).

1. R. J. Johnston, Jr, C. Desplan, Stochastic neuronal cell fate choices. Curr. Opin. Neu- 5. A. Alqadah, Y. W. Hsieh, C. F. Chuang, microRNA function in left-right neu- robiol. 18,20–27 (2008). ronal asymmetry: Perspectives from C. elegans. Front. Cell. Neurosci. 7, 158 2. R. J. Johnston, Jr, C. Desplan, Stochastic mechanisms of cell fate specification (2013). that yield random or robust outcomes. Annu. Rev. Cell Dev. Biol. 26, 689–719 6. A. Alqadah, Y. W. Hsieh, Z. D. Morrissey, C. F. Chuang, Asymmetric development of (2010). the nervous system. Dev. Dyn. 247, 124–137 (2018). 3. R. Losick, C. Desplan, Stochasticity and cell fate. Science 320,65–68 (2008). 7. A. Alqadah, Y. W. Hsieh, R. Xiong, C. F. Chuang, Stochastic left-right neuronal 4. S. L. Reiner, W. C. Adams, Lymphocyte fate specification as a deterministic but highly asymmetry in Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 371, plastic process. Nat. Rev. Immunol. 14, 699–704 (2014). 20150407 (2016).

Alqadah et al. PNAS | December 10, 2019 | vol. 116 | no. 50 | 25145 Downloaded by guest on September 25, 2021 8. Y. W. Hsieh, A. Alqadah, C. F. Chuang, Asymmetric neural development in the 31. D. J. Dickinson, J. D. Ward, D. J. Reiner, B. Goldstein, Engineering the Caenorhabditis Caenorhabditis elegans olfactory system. Genesis 52, 544–554 (2014). elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10, 9. Y. W. Hsieh, A. Alqadah, C. F. Chuang, Mechanisms controlling diversification of ol- 1028–1034 (2013). factory sensory neuron classes. Cell. Mol. Life Sci. 74, 3263–3274 (2017). 32. D. J. Dickinson, B. Goldstein, CRISPR-based methods for Caenorhabditis elegans ge- 10. R. W. Taylor, Y. W. Hsieh, J. T. Gamse, C. F. Chuang, Making a difference together: nome engineering. Genetics 202, 885–901 (2016). Reciprocal interactions in C. elegans and zebrafish asymmetric neural development. 33. D. J. Dickinson, A. M. Pani, J. K. Heppert, C. D. Higgins, B. Goldstein, Streamlined Development 137, 681–691 (2010). genome engineering with a self-excising drug selection cassette. Genetics 200, 1035– 11. E. R. Troemel, B. E. Kimmel, C. I. Bargmann, Reprogramming chemotaxis responses: 1049 (2015). Sensory neurons define olfactory preferences in C. elegans. Cell 91, 161–169 (1997). 34. M. Sato et al., Transportin 1 in the mouse brain: Appearance in regions of neuro- 12. C. F. Chuang, C. I. Bargmann, A Toll-interleukin 1 repeat protein at the synapse genesis, cerebrospinal fluid production/sensing, and circadian clock. J. Comp. Neurol. specifies asymmetric odorant receptor expression via ASK1 MAPKKK signaling. Genes 519 , 1770–1780 (2011). Dev. 19, 270–281 (2005). 35. B. Tursun, L. Cochella, I. Carrera, O. Hobert, A toolkit and robust pipeline for the 13. B. J. Lesch, C. I. Bargmann, The homeodomain protein hmbx-1 maintains asymmetric generation of fosmid-based reporter genes in C. elegans. PLoS One 4, e4625 (2009). in adult C. elegans olfactory neurons. Genes Dev. 24, 1802–1815 36. A. Alqadah, Y. W. Hsieh, C. F. Chuang, A molecular link between distinct neuronal (2010). asymmetries. Cell Cycle 13, 1515–1516 (2014). 14. B. J. Lesch, A. R. Gehrke, M. L. Bulyk, C. I. Bargmann, Transcriptional regulation and 37. A. Alqadah et al., Postmitotic diversification of olfactory neuron types is mediated by stabilization of left-right neuronal identity in C. elegans. Genes Dev. 23, 345–358 differential activities of the HMG-box transcription factor SOX-2. EMBO J. 34, 2574– (2009). 2589 (2015). 15. E. R. Troemel, A. Sagasti, C. I. Bargmann, Lateral signaling mediated by axon contact 38. L. Cochella et al., Two distinct types of neuronal asymmetries are controlled by the and calcium entry regulates asymmetric odorant receptor expression in C. elegans. Caenorhabditis elegans zinc finger transcription factor die-1. Genes Dev. 28,34–43 Cell 99, 387–398 (1999). (2014). 16. S. L. Bauer Huang et al., Left-right olfactory asymmetry results from antagonistic 39. Y. J. Shyu et al., Visualization of protein interactions in living Caenorhabditis elegans functions of voltage-activated calcium channels and the Raw repeat protein OLRN- using bimolecular fluorescence complementation analysis. Nat. Protoc. 3, 588–596 1 in C. elegans. Neural Dev. 2, 24 (2007). (2008). 17. C. Chang, Y. W. Hsieh, B. J. Lesch, C. I. Bargmann, C. F. Chuang, Microtubule-based 40. E. Conti, M. Uy, L. Leighton, G. Blobel, J. Kuriyan, Crystallographic analysis of the localization of a synaptic calcium-signaling complex is required for left-right neuronal recognition of a nuclear localization signal by the nuclear import factor karyopherin asymmetry in C. elegans. Development 138, 3509–3518 (2011). alpha. Cell 94, 193–204 (1998). 18. A. Sagasti et al., The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral 41. D. S. Goldfarb, A. H. Corbett, D. A. Mason, M. T. Harreman, S. A. Adam, Importin signaling decision required for asymmetric olfactory neuron fates. Cell 105, 221–232 alpha: A multipurpose nuclear-transport receptor. Trends Cell Biol. 14, 505–514 (2001). (2004). 19. C. F. Chuang, M. K. Vanhoven, R. D. Fetter, V. K. Verselis, C. I. Bargmann, An innexin- 42. D. Palmeri, M. H. Malim, Importin beta can mediate the nuclear import of an dependent cell network establishes left-right neuronal asymmetry in C. elegans. Cell arginine-rich nuclear localization signal in the absence of importin alpha. Mol. Cell. 129, 787–799 (2007). Biol. 19, 1218–1225 (1999). 20. J. A. Schumacher et al., Intercellular calcium signaling in a gap junction-coupled cell 43. I. V. Aramburu, E. A. Lemke, Floppy but not sloppy: Interaction mechanism of FG- network establishes asymmetric neuronal fates in C. elegans. Development 139, and nuclear transport receptors. Semin. Cell Dev. Biol. 68,34–41 4191–4201 (2012). (2017). 21. A. Alqadah et al., SLO BK potassium channels couple gap junctions to inhibition of 44. R. Bayliss, T. Littlewood, M. Stewart, Structural basis for the interaction between calcium signaling in olfactory neuron diversification. PLoS Genet. 12, e1005654 (2016). FxFG repeats and importin-beta in nuclear trafficking. Cell 102,99– 22. M. K. Vanhoven, S. L. Bauer Huang, S. D. Albin, C. I. Bargmann, The claudin super- 108 (2000). family protein nsy-4 biases lateral signaling to generate left-right asymmetry in 45. G. Cingolani, J. Bednenko, M. T. Gillespie, L. Gerace, Molecular basis for the recog- C. elegans olfactory neurons. Neuron 51, 291–302 (2006). nition of a nonclassical nuclear localization signal by importin beta. Mol. Cell 10, 23. M. Doitsidou, R. J. Poole, S. Sarin, H. Bigelow, O. Hobert, C. elegans mutant identi- 1345–1353 (2002). fication with a one-step whole-genome-sequencing and SNP mapping strategy. PLoS 46. S. J. Lee et al., The structure of importin-beta bound to SREBP-2: Nuclear import of a One 5, e15435 (2010). transcription factor. Science 302, 1571–1575 (2003). 24. A. Harel, D. J. Forbes, Importin beta: Conducting a much larger cellular symphony. 47. M. Putker et al., Redox-dependent control of FOXO/DAF-16 by transportin-1. Mol. Cell Mol. Cell 16, 319–330 (2004). 49, 730–742 (2013). 25. N. Mosammaparast, L. F. Pemberton, Karyopherins: From nuclear-transport mediators 48. M. M. Senchuk et al., Activation of DAF-16/FOXO by reactive oxygen species con- to nuclear-function regulators. Trends Cell Biol. 14, 547–556 (2004). tributes to longevity in long-lived mitochondrial mutants in Caenorhabditis elegans. 26. Y. M. Chook, G. Blobel, Structure of the nuclear transport complex karyopherin- PLoS Genet. 14, e1007268 (2018). beta2-Ran x GppNHp. Nature 399, 230–237 (1999). 49. T. Çagatay,˘ Y. M. Chook, Karyopherins in cancer. Curr. Opin. Cell Biol. 52,30–42 (2018). 27. Y. M. Chook, A. Jung, M. K. Rosen, G. Blobel, Uncoupling Kapbeta2 substrate disso- 50. D. Dormann et al., ALS-associated fused in sarcoma (FUS) mutations disrupt ciation and ran binding. Biochemistry 41, 6955–6966 (2002). Transportin-mediated nuclear import. EMBO J. 29, 2841–2857 (2010). 28. B. J. Lee et al., Rules for nuclear localization sequence recognition by karyopherin 51. V. R. Harley et al., Defective importin beta recognition and nuclear import of the sex- beta 2. Cell 126, 543–558 (2006). determining factor SRY are associated with XY sex-reversing mutations. Proc. Natl. 29. R. D. Finn et al., The pfam protein families database: Towards a more sustainable Acad. Sci. U.S.A. 100, 7045–7050 (2003). future. Nucleic Acids Res. 44, D279–D285 (2016). 52. S. Brenner, The genetics of Caenorhabditis elegans. Genetics 77,71–94 (1974). 30. K. Roayaie, J. G. Crump, A. Sagasti, C. I. Bargmann, The G alpha protein ODR-3 me- 53. G. Minevich, D. S. Park, D. Blankenberg, R. J. Poole, O. Hobert, CloudMap: A cloud- diates olfactory and nociceptive function and controls cilium morphogenesis in based pipeline for analysis of mutant genome sequences. Genetics 192, 1249– C. elegans olfactory neurons. Neuron 20,55–67 (1998). 1269 (2012).

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