Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice - PubMed (original) (raw)
Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice
Vassiliki Fotaki et al. Mol Cell Biol. 2002 Sep.
Abstract
DYRK1A is the human orthologue of the Drosophila minibrain (mnb) gene, which is involved in postembryonic neurogenesis in flies. Because of its mapping position on chromosome 21 and the neurobehavioral alterations shown by mice overexpressing this gene, involvement of DYRK1A in some of the neurological defects of Down syndrome patients has been suggested. To gain insight into its physiological role, we have generated mice deficient in Dyrk1A function by gene targeting. Dyrk1A(-/-) null mutants presented a general growth delay and died during midgestation. Mice heterozygous for the mutation (Dyrk1A(+/-)) showed decreased neonatal viability and a significant body size reduction from birth to adulthood. General neurobehavioral analysis revealed preweaning developmental delay of Dyrk1A(+/-) mice and specific alterations in adults. Brains of Dyrk1A(+/-) mice were decreased in size in a region-specific manner, although the cytoarchitecture and neuronal components in most areas were not altered. Cell counts showed increased neuronal densities in some brain regions and a specific decrease in the number of neurons in the superior colliculus, which exhibited a significant size reduction. These data provide evidence about the nonredundant, vital role of Dyrk1A and suggest a conserved mode of action that determines normal growth and brain size in both mice and flies.
Figures
FIG. 1.
Targeted disruption of mouse Dyrk1A by homologous recombination. (A) Schematic diagram of the Dyrk1A protein. Positions of the catalytic domain and other structural motifs are indicated (NLS, bipartite nuclear localization signal; DH box, consensus sequence rich in aspartate residues; PEST, proline, glutamate, serine, and threonine region; His, histidine repeat; Ser/Thr, serine/threonine repeat; I to XI, subdomains of the catalytic domain). Dashed rectangle indicates the amino acid sequence deleted by gene targeting. (B) Diagram showing the genomic structure of the endogenous wild-type (WT) allele, the targeting vector, and the homologous recombinant (HR) allele. Arrows indicate the transcriptional orientations of the neo gene and the thymidine kinase (tk) gene in the targeting vector. The 5′ flanking probe used for Southern blotting and the expected sizes of the restriction fragments after _Eco_RI digestion of genomic DNA are shown. P1, P2, and P3 indicate the locations of the primers used for PCR analysis (Fig. 2A); P4 and P5 indicate the locations of the primers used for RT-PCR (panel D). (C) Southern blots of _Eco_RI-digested DNA from the wild-type parental ES cell line and three recombinant clones (clones 13, 26, and 40) hybridized with the Dyrk1A probe and a neo probe. (D) (Right) RT-PCR analysis of adult brain tissue with primers external to the targeting event (exons 5 and 9 in panel B) gave rise to a unique product in wild type (+/+) tissue, whereas an additional, shorter product was generated in Dyrk1A heterozygous (+/−) tissue. The same aberrant transcript was also observed in brain and embryonic tissues of all Dyrk1A+/− mice examined. This resulted from aberrant splicing, which led to the deletion of the neo cassette and exon 8 and the introduction of a new stop codon immediately downstream of the targeting event. (Left) Diagram of this alternative transcript.
FIG. 2.
Growth retardation of Dyrk1A−/− embryos. (A) Genotyping of wild-type (Dyrk1A+/+), heterozygous (Dyrk1A+/−), and homozygous (Dyrk1A−/−) embryos by PCR analysis of DNA from embryonic yolk sac tissue. Locations of primers used for amplification of wild-type and targeted alleles are shown in Fig. 1B. (B) Whole-mount in situ hybridization in Dyrk1A+/+ and Dyrk1A−/− E9.5 embryos using a riboprobe containing the deleted Dyrk1A sequence. The absence of signal in the Dyrk1A−/− embryo and its significant size reduction compared to the wild-type embryo can be appreciated. Bars, 1 mm. (C) Western blot analysis of equivalent amounts of protein extracts from Dyrk1A+/+, Dyrk1A+/−, and Dyrk1A−/− embryos with an anti-Dyrk1A specific antibody. The typical cluster of Dyrk1A bands around 90 kDa is shown. (D) Sagittal sections of cresyl violet-stained Dyrk1A+/+ and Dyrk1A−/− embryos at E10.5. The differences between control and homozygous embryos in size and organ development, as well as in the proportions of the brain vesicles, can be appreciated. Bars, 1 mm. (E) (Left panels) Sagittal sections of E10.5 Dyrk1A+/+ and Dyrk1A−/− embryos stained with an anti-β-tubulin antibody (Tuj-1) show a significant decrease in immunostaining for the mutant embryo. Bars, 2 mm. (Right panels) Areas within squares in left panels are amplified, showing fewer immunopositive postmitotic neurons in Dyrk1A−/− embryos than in wild-type embryos. Bars, 200 μm. Abbreviations: atr, common atrial chamber; ba, branchial arch; D, diencephalon; h, heart (truncus arteriosus and bulbus cordis); M, mesencephalon; m, mesodermal tissue; NT, neural tube; op, optic vesicle; ot, otic vesicle; Rh, rhombencephalon; T, telencephalon.
FIG. 3.
Growth retardation and body size reduction of Dyrk1A+/− mice. (A) Comparison between a 1-month-old male wild-type mouse (+/+) and a heterozygous (+/−) littermate indicates the significant body reduction of the Dyrk1A+/− mouse. (B) Somatometric curves of preweaning mice. (Top) Weight gain retardation of Dyrk1A+/− mice early in life. Dyrk1A+/− mice remained underweight throughout life in comparison to their wild-type siblings. (Bottom) Growth retardation during early life in Dyrk1A+/− mice. Each data point represents a mean ± standard deviation. Data points for adult weights represent means of male weights. *, P < 0.001 by repeated-measures ANOVA; for all other data points, P < 0.0001 by repeated-measures ANOVA. (C) Percent decreases in body and organ weights in 3-month-old male Dyrk1A+/− mice relative to those of age-matched Dyrk1A+/+ littermates. Asterisks indicate significant decreases in the body or net organ weight of Dyrk1A+/− mice (n = 4 mice per group; *, P < 0.05 by Student's t test; **, P < 0.005 by Student's t test). Abbreviations: wt, weight; Foreb, forebrain; Hindb, hindbrain and posterior part of mesencephalon.
FIG. 4.
Histological characterization of Dyrk1A+/− brains. (A) Dorsal views of brains of wild type (+/+) and Dyrk1A heterozygous (+/−) adult mice. Arrows point to the mesencephalic tectum, which is particularly decreased in +/− brains. Bars, 2 mm. (B) Nissl-stained sagittal sections of +/+ and +/− brains. Abbreviations: cx, cerebral cortex; H, hippocampus; Hy, hypothalamus; ic, inferior colliculus; Mo, medulla oblongata; ob, olfactory bulb; P, pons; sc, superior colliculus; sp, septum; st, striatum; T, thalamus; Tg, tegmentum. Bars, 2 mm. (C) Coronal sections of the medulla oblongata stained with Nissl. A significant decrease is observed in the Dyrk1A+/− section, although all nuclei (10, dorsal motor; 12, hypoglossal; Gi, gigantocellular reticular; IO, inferior olive; LRt, lateral reticular) are present. Bars, 500 μm. (D) Nissl-stained sagittal sections of +/+ and +/− cerebella. The layers and folia of the Dyrk1A+/− cerebellum are found normal. Bars, 500 μm. (E) Coronal sections of parvalbumin-immunostained brains reveal normal lamination of the Dyrk1A+/− cerebral cortex and hippocampus. Bars, 300 μm. (F) GFAP immunostaining in PD14 coronal brain sections shows increased GFAP immunoreactivity in Dyrk1A+/− mice. 4V, 4th ventricle. Bars, 75 μm.
FIG. 5.
Alterations in the mesencephalic tectum of Dyrk1A+/− mice. Shown are coronal sections of the SC of wild type (+/+) and Dyrk1A heterozygous (+/−) adult mice immunostained with calbindin (A) or calretinin (B). Dashed lines mark the thickness of the SGS and SO. The size reductions of these superficial layers in Dyrk1A+/− mice can be appreciated. SGI, stratum griseum intermedium. Bars, 100 μm.
FIG. 6.
Preweaning neurobehavioral analysis. Bars represent mean PDs for the emergence of physical landmarks or the achievement of mature responses in reflexological and behavioral tasks. **, P < 0.001 by Student's t test; ***, P < 0.0001 by Student's t test. Error bars, standard errors. Inf, inferior.
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