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Corresponding author: Oliver Hobert, College of Physicians and Surgeons, 701 W. 168th St., HHSC 724, New York, NY 10032., or38{at}columbia.edu (E-mail)
Communicating editor: B. J. MEYER
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.
TRISOMY of chromosome 21, or Down syndrome, is the most frequent chromosomal abnormality in human infants that come to term. Besides manifesting a characteristic set of facial and physical features, heart defects, and abnormalities in the immune and endocrine systems, patients with Down syndrome have deficits in spatial memory and difficulty in converting short-term to long-term memories (
The first vertebrate member of the DYRK family was originally identified in a PCR screen for protein kinases (
In metazoan animals, the only DYRK/minibrain family member for which mutant alleles exist is the Drosophila minibrain gene. Here, we describe the expression pattern and loss-of-function alleles of all three DYRK/minibrain family members in the nematode Caenorhabditis elegans. In contrast to Drosophila minibrain, we observe no obvious morphological defects in mbk-1 mutants, but find that another DYRK/minibrain family member, mbk-2, is required for viability. In an attempt to model the cellular consequences of DYRK1A overexpression that are observed in Down syndrome patients, we analyzed the effects of providing extra copies of the worm ortholog of DYRK1A and describe dosage-sensitive and reversible defects in the processing of acute sensory information.
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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cDNAs:
A full-length mbk-1 cDNA was amplified with SL1 and gene-specific primers. The structure of the cDNA is similar to the predicted T04C10.1 gene, with the exception of an incorrect first exon predicted in T04C10.1. The mbk-2 locus generates two messages, a long form (mbk-2L) and a short form (mbk-2S) that uses an internal start site from an alternatively spliced exon. Both splice forms are represented in expressed sequence tag (EST) clones (a gift from Y. Kohara) that have been completely sequenced. Similarly, hpk-1 full-length cDNA clones are represented in Y. Kohara's EST collection.
DNA constructs:
Constructs are shown schematically in Fig 1.
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mbk-1:
To build mbk-1::gfp, the mbk-1 genomic locus, including 7 kb of 5' noncoding sequence and all exons and introns, was amplified by Expand long-template PCR (Boehringer Mannheim, Indianapolis). The PCR product was cloned in frame with gfp in the promoterless vector pPD95.75 (from A. Fire), generating pBR104. To build mbk-1(YA)::gfp, the QuickChange kit (Stratagene, La Jolla, CA) was used to modify pBR104 with primers YAs (5'-ctggacaccgaatcgcccaggccattcagtcgagattctatcg) and YAas (5'-cgatagaatctcgactgaatggcctgggcgattcggtgtccag), generating pBR113. To build mbk-1(pk1389)::gfp, the mbk-1 locus was amplified from homozygous mbk-1(pk1389) animals by Expand long-template PCR (Boehringer Mannheim). Following the cloning and sequencing of this PCR product, a 4.0-kb BglII-Asp718 fragment was subcloned into pBR104, generating pBR177. To build pgcy-10::mbk-1, the gcy-10 promoter (
mbk-2: To build pmbk-2L::gfp, 8 kb of 5' noncoding sequence was amplified by Expand long-template PCR (Boehringer Mannheim) and cloned into pPD95.77 (from A. Fire), generating pBR126. To build mbk-2S::gfp, Expand long-template PCR (Boehringer Mannheim) was used to amplify exons 7–11 of the mbk-2 locus and fused in frame with gfp in pPD95.77 (from A. Fire), generating pBR138. This construct uses intron 6 as the 5' noncoding sequence. pmbk-2L::gfp and mbk-2S do not contain the full mbk-2 genomic locus, and this may result in an artifactual or incomplete expression pattern. To build phsp16-2::mbk-2L::gfp, full-length mbk-2L cDNA was fused with gfp and inserted into the heat-shock vector pPD49.79 (from A. Fire), generating pBR169.
hpk-1: To build a translational fusion between HPK-1 and green fluorescent protein (GFP), the full-length hpk-1 genomic locus, including 4 kb of 5' noncoding sequence and all exons and introns, was amplified by Expand long-template PCR (Boehringer Mannheim) and cloned into the promoterless vector pPD95.77 (from A. Fire), generating pBR132.
Transgenic and mutant strains:
The strains are as follows:
All expression constructs were injected at 50 ng/µl. pNC4.21 [unc-4(+)] and unc-122::gfp were injected at 50 ng/µl. pRF4 [rol-6(d)] was injected at 100 ng/µl. Integrated lines were obtained using a protocol described at http://cpmcnet.columbia.edu/dept/gsas/biochem/labs/hobert/integration_protocol.html.
Isolation of deletions in the mbk-1, mbk-2, and hpk-1 loci:
PCR screening of a chemically induced deletion library was done as previously described (
RNAi:
mbk-2 dsRNA was delivered by bacterial feeding as previously described (
Behavioral assays:
Chemotaxis assays toward volatile odorants were performed as described (
Scoring neuroanatomy:
AWC morphology was observed by crossing kyIs140, a chromosomally integrated str-2::gfp construct (
DAPI staining:
4',6-Diamidino-2-phenylindole (DAPI) staining on gfp-expressing transgenic lines was done by placing animals in a drop of water on a coverslip, letting the water evaporate, adding a drop of acetone, letting the drop evaporate, drying for 20 min, and then adding 1 µg/ml DAPI in M9 medium.
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Identification of DYRK/minibrain-like genes in C. elegans:
Through sequence homology searching of the complete C. elegans genome sequence, we identified two C. elegans genes with close homology to the DYRK/mini-brain family, termed mbk-1 and mbk-2 (for minibrain- kinase), and one gene, hpk-1 (named after its vertebrate homologs, homeodomain-interacting protein kinase 1-3), with a more distant homology (Fig 2A). mbk-1 and mbk-2 both carry the characteristic DYRK family signature motifs in their kinase domain, as well as a DH-box, a conserved sequence motif preceding the catalytic domain (
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Expression and subcellular localization of DYRK/minibrain-like genes in C. elegans:
To determine the sites of expression and subcellular localization of DYRK/minibrain-like genes, we fused gfp in frame with the respective genomic loci (Fig 1). The mbk-1::gfp construct included 7 kb of 5' noncoding sequence and all of the exons and introns present in the endogenous mbk-1 locus. The mbk-1::gfp gene product is expressed in all somatic cells and primarily localizes to nuclei (Fig 3), similar to the reported expression and subcellular localization of human DYRK1A (
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In contrast to mbk-1, mbk-2::gfp reporter constructs were expressed in subsets of tissues, including the nervous system, body wall muscle, and the pharynx (Fig 4, A–C). To determine the subcellular localization of MBK-2 protein, we fused a cDNA—encoding the longer splice form of mbk-2 (see Fig 1 and MATERIALS AND METHODS)—to gfp and expressed it under control of a ubiquitously expressed promoter (Fig 1). Unlike MBK-1::GFP, but like its vertebrate ortholog DYRK2 (
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A translational HPK-1::GFP reporter construct (Fig 1) was broadly expressed during embryogenesis and localized to moving nuclear puncta (Fig 4, E–H). In adult animals, the number, intensity, and movement of HPK-1-GFP puncta are greatly reduced. Although we have not further examined the identity of these puncta, the observation that the three vertebrate homologs of HPK-1, HIPK1–3, stably bind and phosphorylate nuclear transcription factors (
Taken together, the distinctive subcellular localization patterns described here for the individual C. elegans DYRK/minibrain family members reveal a striking similarity to their presumptive vertebrate orthologs and, together with the primary sequence similarity, suggests that vertebrate and C. elegans DYRK/minibrain proteins fulfill similar functions.
Providing extra copies of mbk-1 causes dosage-sensitive olfactory defects:
Because human DYRK1A overexpression has been implicated in the behavioral defects of Down syndrome, we wanted to examine the consequences of overexpression of the DYRK1A ortholog mbk-1 on C. elegans behavior. Toward this end, a multicopy array derived from a subcloned amplicon of the mbk-1 genomic locus was integrated into the genome of wild-type worms, giving rise to multiple independent lines that harbor additional copies of mbk-1 (see Fig 1 for constructs). An appended gfp tag allowed us to monitor ectopic mbk-1 expression, which we observed broadly throughout the whole animal (Fig 3). In light of the multicopy nature of stably transmitted DNA arrays in C. elegans (
All of the transgenic lines that contain extra copies of mbk-1, here on referred to as mbk-1(gf) animals, appeared normal in respect to overall morphology, life span, dauer formation, thermotaxis, mechanosensation, and chemotaxis toward water-soluble attractants (data not shown). mbk-1(gf) animals display no defects in locomotory rate or reversal behavior (data not shown). However, olfaction toward several volatile chemoattractants was markedly disrupted in mbk-1(gf) animals (Fig 5). Comparable defects were observed with chromosomally integrated lines that contain extra copies of either wild-type mbk-1 or a mutated version of mbk-1 in which the kinase activity was potentially hyperactivated (Y-to-E change in kinase domain; data not shown). mbk-1(gf) defects are apparent throughout a wide range of odor concentrations.
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The olfactory-defective phenotype was sensitive to gene dosage, since animals heterozygous for the integrated mbk-1 array display intermediate odortaxis defects. As a control, we provided extra copies of mbk-1, again under the control of its own promoter and tagged with gfp to monitor expression, but containing an inactivated kinase domain (Fig 1). We found that worms containing integrated copies of the mbk-1(YA) genomic construct showed no defects in odortaxis (Fig 5A). Additional copies of mbk-2 also produced no olfaction defect (Fig 6B). All ectopic expression constructs yielded comparable levels of protein expression as assessed by the added GFP tag. The olfaction phenotype thus requires the kinase activity of mbk-1, is specific to mbk-1, and is sensitive to gene dosage.
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Attractive olfactory responses in C. elegans are largely mediated by two bilaterally symmetric pairs of ciliated sensory neurons, called AWA and AWC (
The cellular specificity of odortaxis defects induced by extra copies of mbk-1 agrees with our observation that mbk-1(gf) animals are indistinguishable from wild-type animals in all other behaviors tested and suggests that mbk-1 acts selectively within specific cellular contexts rather than affecting neuronal signaling in a broad and unspecific way.
mbk-1 can act autonomously in sensory neurons:
To elucidate the cellular context in which extra copies of mbk-1 can affect olfactory behavior, we added extra copies of mbk-1 specifically in olfactory neurons using the gcy-10 promoter, which is active in the AWC and AWB chemosensory neurons, as well as in the pharyngeal interneuron I1 (
mbk-1 can act reversibly and acutely in differentiated neurons:
Given that odortaxis defects arise from increasing mbk-1 gene dosage, an important issue is determining the temporal window of mbk-1 function: Is the increased gene dosage altering development or the function of already differentiated cells? To address this issue, we followed several approaches: First, we tested whether AWC development is affected in mbk-1(gf) mutants by assessing AWC generation, proliferation, or anatomy using str-2::gfp (
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Intriguingly, the effects of providing extra copies of mbk-1 overexpression were reversible, since adult animals tested 24 hr after mbk-1 induction displayed normal olfaction (Fig 6B; note that decrease in mbk-1 expression could be monitored with the attached gfp tag). These results indicate that extra copies of mbk-1 interfere with the acute function, but not with the formation or differentiation, of the olfactory neurons and that the phenotypes resulting from overexpressed mbk-1 can be reversed by reducing mbk-1 levels.
Loss-of-function analysis of mbk-1:
The defects caused by providing additional copies of mbk-1 prompted us to elucidate the mbk-1 loss-of-function phenotype. We isolated a mbk-1 loss-of-function allele, pk1389, through PCR screening of a C. elegans deletion library. pk1389 is a likely null allele since it eliminates exons 2–6 of mbk-1. Exon splicing around this deletion would lead to a frameshift and to a premature stop in the message or to a deletion of the entire kinase domain (Fig 1 and MATERIALS AND METHODS). To confirm this notion, we introduced the pk1389 deletion into the context of the translational mbk-1::gfp construct (Fig 1) and found that transgenic animals expressing this construct show no gfp expression (data not shown).
mbk-1(pk1389) homozygous mutant animals are viable and show no obvious morphological abnormalities. We examined several aspects of nervous system function in mbk-1 mutants, including the behavior of mbk-1 mutants in response to various sensory inputs (mechano- and chemosensory), and detailed aspects of its locomotory behavior. We observed no obvious defects under standard conditions (data not shown). We observed slight but significant defects in attraction to low doses of olfactory cues, but since we were not able to rescue this phenotype through expression of mbk-1 from extrachromosomal arrays, we could not conclude that these defects are indeed due to loss of mbk-1 (data not shown; these rescue experiments may have failed because of an inability to supply precisely the right amount of mbk-1).
We examined nervous system architecture of mbk-1 mutant animals in more detail. We visualized a total of 18 individual head and tail sensory neurons and their axonal anatomy in the main head ganglia of C. elegans both with gfp reporter genes that label individual cells and with the fluorescent dye DiI (
Isolation of loss-of-function alleles in other DYRK1A/minibrain family members:
Since mbk-1 may act redundantly with other DYRK1A/minibrain family members to affect C. elegans nervous system development or function, we isolated mutations in the only other two DYRK1A/ minibrain-related genes in the C. elegans genome, namely mbk-2 and hpk-1 (Fig 1 and Fig 2). Due to their deletion of the respective kinase domains, the mbk-2 and hpk-1 mutant alleles constitute likely null alleles (Fig 1).
While hpk-1 null mutant animals are viable and appear indistinguishable from wild type (data not shown), mbk-2(pk1427) homozygous animals display 100% penetrant maternal-effect embryonic lethality. The maternal-effect lethality is caused by defects in spindle positioning and cytokinesis in the early embryo (J. PELLETTIERI and G. SEYDOUX, personal communication). In addition, mbk-2 homozygous animals derived from a heterozygous parent appear sick and grow poorly. To independently confirm the linkage of the lethality with the mbk-2 locus, we inactivated mbk-2 by RNAi (
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our analysis of the C. elegans homologs of the DYRK/minibrain family of protein kinases reveals strong patterns of similarity in expression and subcellular localization of these proteins across phylogeny. Like their vertebrate orthologs, all three DYRK/minibrain-like genes are broadly expressed. MBK-1, like its vertebrate ortholog DYRK1A, localizes to the nucleus while the closely related MBK-2, like its vertebrate ortholog DYRK2, is predominantly cytoplasmic and excluded from the nucleus. HPK-1, like its vertebrate homolog HIPK2, is localized to subnuclear puncta. However, interspecies comparisons in mutant phenotypes reveal a strong dissimilarity. While the Drosophila minibrain locus affects neuroblast proliferation and hence brain size (
The most important conclusions from our functional analysis of DYRK1/minibrain family genes derive from providing extra copies of the mbk-1 gene. First, increases in mbk-1 expression produce functional olfactory defects without apparently interfering with either neuronal proliferation or neuronal differentiation. Second, the phenotypes resulting from providing extra copies of mbk-1 can be specifically induced in adult animals and are thus acute rather than developmental defects. Third, the defects can be reversed by restoring mbk-1 to normal levels of expression. Fourth, extra copies of the ubiquitously expressed mbk-1 gene specifically disrupt olfactory behavior but no other behaviors tested. This apparent specificity suggests that additional copies of mbk-1 do not cause an unspecific disruption of signaling or sickening of a neuron, but act in a cellular-context-dependent manner.
We conclude that aberrant expression levels of mbk-1 interfere with acute sensory processing in olfactory neurons. This finding is relevant since it is the additional copy of the vertebrate mbk-1 ortholog DYRK1A that causes neurological defects either in humans or in mouse models. Our experiments with the temporal regulation and reversibility of mbk-1 expression phenotypes may be taken as an indication that human DYRK1A overexpression disrupts the function of terminally differentiated neurons. Moreover, the reversibility of defects observed upon the reversion of mbk-1 expression levels back to normal suggests that therapies aimed at the reduction of DYRK1A expression or activity may provide a viable therapeutic approach for the neurological defects of Down syndrome.
| ACKNOWLEDGMENTS |
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We thank Y. Kohara (National Institute for Genetics, Mishima, Japan) for cDNA clones, A. Fire (Carnegie Institution, Baltimore) for vectors, members of the worm community for plasmids and strains, G. Seydoux for communicating unpublished results, and the Caenorhabditis Genetics Center for the nematode strains used in this study. We are grateful to Iva Greenwald, Piali Sengupta, and members of the Kandel and Hobert labs for helpful comments during the preparation of this manuscript and to Kelvin Pau and Ephraim Tsalik for help with performing several behavioral assays. This work was funded by the Howard Hughes Medical Institute, National Institutes of Health postdoctoral fellowship 5 F32 NS11114 to W.B.R., The Netherlands Organization for Scientific Research, and the Klingenstein, Rita Allen, Hirschl, Sloan, McKnight, and Whitehall Foundations (O.H.).
Manuscript received June 18, 2002; Accepted for publication November 5, 2002.
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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