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Link to original content: http://pubmed.ncbi.nlm.nih.gov/31767767/
A universal transportin protein drives stochastic choice of olfactory neurons via specific nuclear import of a sox-2-activating factor - PubMed Skip to main page content
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. 2019 Dec 10;116(50):25137-25146.
doi: 10.1073/pnas.1908168116. Epub 2019 Nov 25.

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

Amel Alqadah  1 , Yi-Wen Hsieh  1 , Rui Xiong  1 , Bluma J Lesch  2 , Chieh Chang  1 , Chiou-Fen Chuang  3
Affiliations

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

Amel Alqadah et al. Proc Natl Acad Sci U S A. .

Abstract

Stochastic neuronal cell fate choice involving notch-independent mechanisms is a poorly understood biological process. The Caenorhabditis elegans AWC olfactory neuron pair asymmetrically differentiates into the default AWCOFF and induced AWCON subtypes in a stochastic manner. Stochastic choice of the AWCON subtype is established using gap junctions and SLO BK potassium channels to repress a calcium-activated protein kinase pathway. However, it is unknown how the potassium channel-repressed calcium signaling is translated into the induction of the AWCON subtype. Here, we identify a detailed working mechanism of how the homeodomain-like transcription factor NSY-7, previously described as a repressor in the maintenance of AWC asymmetry, couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype through the identification of a unique imb-2 (transportin 1) allele. imb-2 loss-of-function mutants are not viable; however, we identify a viable imb-2 allele from an unbiased forward genetic screen that reveals a specific role of imb-2 in AWC olfactory neuron asymmetry. IMB-2 specifically drives nuclear import of NSY-7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box transcription factor SOX-2 for the specification of the AWCON subtype. This study provides mechanistic insight into how NSY-7 couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype. Our findings also provide structure-function insight into a conserved amino acid residue of transportins in brain development and suggest its dysfunction may lead to human neurological disorders.

Keywords: NSY-7; asymmetry; sox-2; stochastic choice; transportin 1.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
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) Expression of AWCON 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.
Fig. 2.
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.
Fig. 3.
Fig. 3.
imb-2/transportin 1 acts cell autonomously to promote the AWCON subtype. (A) Images of imb-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] and 50 μm [iii].) Asterisks indicate nonadult animals. (B) Images of imb-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) Images of imb-2::mNG knock-in expression at a higher level in AWCON than in AWCOFF in a L1 animal (dorsal view). AWCON was marked by str-2p::TagRFP and ceh-36p::myrTagRFP, while AWCOFF 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), and imb-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.
Fig. 4.
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 nonfluorescent fragments of Venus, at the L1 stage. (Scale bar, 5 μm.) VN, VN173 (Venus 1–172); VC, VC155 (Venus 155–238).
Fig. 5.
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) Quantification 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 concentrations (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.
Fig. 6.
Fig. 6.
Model of imb-2 function in AWC asymmetry. (A) In wild-type animals, 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 nucleus to activate sox-2 expression thereby inducing the AWCON identity. (B) In imb-2(vy10) mutants, NSY-7 fails to enter the nucleus, leading to loss of sox-2 expression and a 2AWCOFF phenotype. Gray, less active or inactive molecules or expression.
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