Systematic Functional Characterization of Human 21st Chromosome Orthologs in Caenorhabditis elegans

Abstract

Individuals with Down syndrome have neurological and muscle impairments due to an additional copy of the human 21st chromosome (HSA21). Only a few of ∼200 HSA21 genes encoding proteins have been linked to specific Down syndrome phenotypes, while the remainder are understudied. To identify poorly characterized HSA21 genes required for nervous system function, we studied behavioral phenotypes caused by loss-of-function mutations in conserved HSA21 orthologs in the nematode Caenorhabditis elegans We identified 10 HSA21 orthologs that are required for neuromuscular behaviors: cle-1 (COL18A1), cysl-2 (CBS), dnsn-1 (DONSON), eva-1 (EVA1C), mtq-2 (N6ATM1), ncam-1 (NCAM2), pad-2 (POFUT2), pdxk-1 (PDXK), rnt-1 (RUNX1), and unc-26 (SYNJ1). We also found that three of these genes are required for normal release of the neurotransmitter acetylcholine. This includes a known synaptic gene unc-26 (SYNJ1), as well as uncharacterized genes pdxk-1 (PDXK) and mtq-2 (N6ATM1). As the first systematic functional analysis of HSA21 orthologs, this study may serve as a platform to understand genes that underlie phenotypes associated with Down syndrome.

Keywords: Down syndrome; neurological; neuromuscular; synaptic.

Figure 1

Representation of human 21st chromosome genes in C. elegans. (A) HSA21 encodes a conservative estimate of 213 protein-coding genes. Excluding the 48 genes predicted to encode keratin on HSA21 (dark gray), over half of the remaining 165 genes have predicted orthologs in C. elegans as identified by OrthoList. All 85 putative orthologs in worm (light gray and blue) with an available RNAi clone were tested in the RNAi viability screen (Table S1). (B) Representation of InParanoid-predicted orthologs with their human to worm relationship (shades of blue). We tested all predicted InParanoid orthologs except those within a many-to-many relationship with RNAi for viability and mutants for behavioral and aldicarb assays (red).

Figure 2

Screen for radial dispersion defects. Histograms show the mean displacement ± SEM from start over 10 min for each worm during a radial dispersion assay. (A) Three mutants (green) showed substantial reductions in dispersal relative to wild type (black). *** P < 0.001, n > 30. (B) Reductions in dispersal were rescued for the cysl-2 and mt-1 mutants (green) by extrachromosomal expression of the wild-type genes (blue). Wild-type performance shown for comparison (black). * P < 0.05, ** P < 0.01, *** P < 0.001, n > 30.

Figure 3

Screen for exploration defects. Histograms show the mean percentage of the assay plate traversed (percent of squares entered ± SEM) by individual worms over a 16-hr exploration assay. (A) Five mutants (green) covered significantly less area than wild type (black). *** P < 0.001, n > 15. (B) Reductions in exploration were rescued in the eva-1, C24H12.5, mtq-2(tm3565), and pdxk-1 mutants (green) by extrachromosomal expression of each of the wild-type genes (blue). Wild-type performance shown for comparison (black). * P < 0.05, ** P < 0.01, *** P < 0.001, n >15.

Figure 4

Screen for pumping defects. Histograms showing mean pharyngeal pumps per minute (±SD) in the presence of standard bacterial food. (A) Seven mutants (green) showed significant defects in pharyngeal pumping relative to wild type (black). *** P < 0.001, n > 30. (B) Mutants with pharyngeal pumping defects mtq-2(tm3565), pad-2, ncam-1, and cle-1 (green) were rescued by extrachromosomal expression of the wild-type genes (blue). We did not observe rescue of B0024.15. * P < 0.05, *** P < 0.001, n > 30.

Figure 5

Screen for aldicarb resistance. We measured the full 180-min time course to paralysis for 28 strains including wild type (WT) and hypersensitive control (dgk-1) in 1-mM aldicarb. Significant difference in paralysis rate was determined from WT with a two-way ANOVA using Dunnett’s correction for multiple comparisons. No mutants showed significant hypersensitivity. (A) Mean percent (±SEM) of animals moving on aldicarb over minutes for WT, positive control, and mutants that were significantly different from WT: pdxk-1(gk855208), mtq-2(tm3565), mtq-2(ok3740), and unc-26(e345). n > 3. (B) Mean percent of (±SEM) of animals moving on aldicarb at the 180 min time point for all mutants tested including those (green) that were significantly different from WT (black). Zero indicates that no animals were moving. *** P < 0.001.

Figure 6

Response of deletion and putative loss-of-function mutants to aldicarb and levamisole. Time course to paralysis on 1 mM aldicarb or 800 μM levamisole for aldicarb-resistant mutants identified in screen (n > 3 trials, ∼25 worms per trial). Wild-type controls were assayed in parallel. (A) Two independent alleles of mtq-2 (green) displayed significant resistance to aldicarb, P < 0.001. Two of two rescue lines of mtq-2(tm3565) (blue) displayed significant sensitivity from the mutant background, P < 0.05. (B) Two independent alleles of mtq-2 displayed sensitivity to levamisole. (C). pdxk-1(gk855208) displayed significant resistance to aldicarb, P < 0.001. One of two rescue lines of pdxk-1(gk855208) (blue) was significantly improved from the mutant background, P < 0.01. (D) pdxk-1(gk855208) displayed hypersensitivity to levamisole relative to wild type, P < 0.001. (E) unc-26(e345) displayed significant resistance to aldicarb, P < 0.001 and (F) hypersensitivity to levamisole, P < 0.001.

Figure 7

Expression pattern of select genes. Fluorescent micrographs of all HSA21 ortholog hits for which expression patterns were unknown. (A1–A3) A transcriptional reporter for mtq-2 expressed mCherry throughout the adult nervous system including in head (A3). Bright field image shown in (A1) for reference. Scale bar, 100 μm. Dual color images show coexpression of mtq-2 reporter in cholinergic (arrowheads, A4) but not in GABAergic neurons (A5) in the ventral nerve cord. Scale bar, 50 μm. (B1–B3) A transcriptional reporter for pdxk-1 expressed mCherry throughout the adult nervous system including in head (B3). Bright field image shown in B1 for reference. Scale bar, 100 μm. Dual color images show coexpression of pdxk-1 reporter in GABAergic neurons (B5) but not cholinergic neurons (B4) in the ventral nerve cord. Scale bar, 50 μm. (C) A transcriptional reporter for dnsn-1 expressed mCherry widely in developing embryo. Scale bar, 50 μm. (D) A transcriptional reporter for cysl-2 expressed mCherry throughout body-wall and vulval muscle. Scale bar, 100 μm.