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First published online 25 July 2007
doi: 10.1242/dev.005884

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Mice lacking sister chromatid cohesion protein PDS5B exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome

¹ Departments of Genetics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
² Departments of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
³ Departments of Medicine and Renal division, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
⁴ Departments of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
⁵ Departments of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
⁶ Departments of HOPE Center for Neurological Disorders, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Expression of Pds5B and generation of Pds5B-deficient mice. (A) Quantitative RT-PCR analysis of Pds5B mRNA levels in adult mouse tissues normalized to 18S RNA. (Pr, prostate; Te, testis, St, stomach; Lu, lung; Sp, spleen; He, heart; Br, brain; Li, liver; Ki, kidney; Th, thymus). Error bars represent ± s.e.m. (B,C,D) Radioactive in situ hybridization (ISH) using 35S-CTP-labeled probes was performed using WT adult testis (B), adult brain (C), and E13.5 embryo (D). Silver grains indicate Pds5B mRNA. The green background is the Eosin counterstain visualized by darkfield microscopy. Note the high signal at the periphery of the seminiferous tubules (ST) consistent with Pds5B expression in immature germ cells (arrows in B), in hippocampus (arrow in C) and dentate gyrus (arrowhead in C), and wide spread expression in the E13.5 mouse embryo (D). In D, the sagittal sections are probed with antisense (AS, left) and sense control (S, right) probes. (E) Schematic representation of homologous recombination between Pds5B gene (top) and the targeting construct (middle) to give the mutant Pds5B allele (bottom). The coding region of the second exon of Pds5B is replaced by a ß-gal/PGK-Neo (ßGal Neo) cassette. The floxed PGK-Neo cassette was later excised by mating to ß-actin Cre mice. K, KpnI; N, NheI; S, SacI. Black bars with numbers indicate exons and P1-P3 indicate positions of primers used for genotyping. (F) Southern blot analysis depicting successful targeting of the Pds5B locus using genomic DNAs from tails of wild-type (+/+), heterozygous (+/-), homozygous (-/-) mice. The DNA samples were digested with NheI and hybridized with the radiolabeled DNA probe, indicated in E. (G) PCR genotyping analysis of the Pds5B mutation was performed with primers P1, P2, and P3 indicated in E. (H) Relative amounts of Pds5B mRNA from wild-type (+/+), heterozygous (+/-) and homozygous (-/-) embryonic E14.5 brains using qRT-PCR normalized to GAPDH mRNA level. Error bars represent the s.e.m. *P<0.001, Student's unpaired t-test. (I) Western blot on E14.5 forelimb tissue lysates was probed with antibodies to PDS5B and ß-tubulin. Note undetectable PDS5B protein in Pds5B-/- lysate.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Pds5B deficiency results in growth retardation, abnormal skeletal patterning and cleft palate. (A) The weight of Pds5B-/- P0 mice is lower than that of wild-type littermates (WT, n=43 and Pds5B-/- mice, n=35). Error bars represent s.e.m. *P<0.001, Student's unpaired t-test. (B,C) Morphology of wild-type and mutant mice. (B) Note short stature, facial dysmorphism, short limbs (arrow), short snout (arrowhead), and small head in Pds5B-/- compared to wild-type P0 mice. (C) Short mandible (arrow) in Pds5B-/- neonate (right) compared to wild-type (left). (D-I) Alizarin Red S and Alcian Blue staining of newborn mouse skeleton. Red indicates bone and blue indicates cartilage. (D) Mandibles; (E) ulna, radius and humerus of forelimbs; (F) scapula of forelimbs. Bone lengths (square bracket) are indicated by numbers (lengths in mm ± s.d.). Note the length of mandible, ulna, radius and humerus is significantly shorter in Pds5B-/- compared to wild-type. Sample sizes for each genotype range from 18 to 34. P<0.001, Student's unpaired t-test. (G) Sternum. Arrows, unfused ossification centers; arrowheads, delayed or missing ossification centers. (H) Ribs. The arrow points to the C7-T1 fusion. (I) Thoracic (T) and lumbar (L) vertebrae. The arrow points to the unfused L3 vertebra and arrowheads point to the hypoplastic 13th ribs in Pds5B-/- neonate. (J,K) Complete cleft palate (arrow) and thin upper-lip (arrowhead) of a Pds5B-/- neonate (K) compared with wild-type control (J). (L,M) Alizarin Red S and Alcian Blue staining of neonate skulls. The edges of palatal bones are marked by dashed lines. Posterior parts of the palatal bones in Pds5B-/- mutant failed to meet at the midline (arrows, M), compared with wild-type littermate (L). (N-S) Palatogenesis defects illustrated using H&E stained coronal sections of wild-type and Pds5B-/- E12.5, E14.5, and E18.5 embryos. Wild-type (N-P); Pds5B-/- (Q-S). ps, palatal shelf; n, nasal bone; to, tongue. Arrow in S points to the unfused palatal shelves. Scale bars:1 mm.

View larger version (115K): [in this window] [in a new window]   Fig. 3. Congenital heart defects in Pds5B-/- mice mimic those observed in CdLS patients. (A,B) Sections of wild-type mouse hearts demonstrating (A) the intact ventricular septum and relationships of the aortic, tricuspid and mitral valves, and (B) an intact atrial septum. (C-F) Sections of mutant mouse hearts showing: (C) a large perimembranous VSD with the aorta riding over both ventricles; (D) a secundum ASD; (E) a muscular VSD; (F) a common atrioventricular canal defect. Note the single atrioventricular valve and the common defect of the atrial and ventricular septum. Arrows indicate the respective defects. AoV, TV, MV, aortic, tricuspid and mitral valve; RA, LA, right, left atrium; RV, LV, right, left ventricle. Scale bar: 1 mm. (G) Summary of congenital heart defects in Pds5B-/- mice. P<0.001 is calculated by a Z-test for significant differences in total incidence between WT and Pds5B homozygotes.

View larger version (81K): [in this window] [in a new window]   Fig. 4. Pds5B-/- mice display multiple defects in the peripheral nervous system. (A) Summary of carotid nerve and superior cervical ganglion (SCG) abnormalities in Pds5B-deficient mice. SCG AML, SCG delayed migration or abnormal localization; TCN, thin carotid nerve; CNM, carotid nerve missing. *P<0.05 and **P<0.001 determined by a Z-test for significant differences in incidence between WT and Pds5B homozygotes. (B-G) TOH whole-mount staining of sympathetic neurons of neonate mice. (B,C) Abnormal caudal location of SCG in Pds5B-/- mice compared to wild-type mice (SCG, arrows). Dashed circle indicates the expected normal location of SCG (C). Note the severe reduction or absence of carotid nerves in Pds5B-/- (E,G) compared to WT mice (D,F). Carotid nerves are indicated by arrows in D and E, and by two dashed lines in F and G. ey, eye; co, cochlea. Scale bars: 400 µm (B-E); 200 µm (F,G). (H,I) E12.5 enteric neurons in the distal bowel of whole gut demonstrated by TuJ1 immunohistochemistry (H, wild type; I, Pds5B-/-). *, ileocecal junction. Note that the enteric neuron migration wave front did not pass the ileocecal junction in Pds5B-/- mice (I). Scale bars: 10 µm (H,I). (J,K) TuJ1 staining of P0 paraffin sections of distal colon from wild-type (J) and Pds5B-/- mice (K), showing the lack of clustered enteric neuron cell bodies in Pds5B-/- mice. Scale bars: 20 µm. (Insets) High magnification of TuJ1 staining of P0 distal colons. Scale bars: 10 µm. Arrowheads point to the clustered enteric neuron cell bodies. (L,M) Tuj1 whole-mount staining of E18.5 guts from wild-type mice (L) and Pds5B-/- (M). Wild-type mice show normal formation of enteric plexus with clustered neuronal cell bodies (arrowheads) in the mid-colon, whereas Pds5B-/- mice lack enteric neuron cell bodies and show only a few thin nerve fibers (arrows). Scale bar: 100 µm. (N) Schematic representation of ENS defects in Pds5B-/- mice [E12.5: Pds5B-/- (n=7) and WT (n=9); P0: Pds5B-/- (n=12) and WT (n=13)]. The scale at the top corresponds to the percentage of the respective intestinal segment (small or large intestine) successfully colonized by neurons.

View larger version (77K): [in this window] [in a new window]   Fig. 5. Pds5B-deficient mice manifest germ cell defects. GCNA1 staining of germ cells of WT (A) and Pds5B-/- (B) neonatal ovaries, and WT (C) and Pds5B-/- (D) neonatal testes show reduced germ cells in Pds5B-/- mice. Arrows point to empty testicular cords in D. (E,F) GCNA1 immunohistochemical analysis of E16.5 germ cells in WT (E) and Pds5B-deficient (F) testes shows a reduction in germ cells during embryogenesis. (G,H) GCNA1 staining of two-week testes transplants of E16.5 wild-type (G) and Pds5B-/- mice (H). In contrast to the presence of germ cells in the periphery of WT seminiferous tubules (G), note complete loss of germ cells in Pds5B-/- testis explant (H). *Non-specific auto-fluorescence signal; red, GCNA1; blue, bisbenzimide. (I) Quantitative analysis of germ cells in E16.5 testes of WT and Pds5B-deficient mice (n=4 for each genotype). Error bars represent SEM. *P<0.001, Student's unpaired t-test. (J) Proliferation index measured by BrdU incorporation in GCNA1-positive germ cells at E12.5 (n=3 for each genotype and 100 GCNA1-positive cells were counted for each animal). Error bars represent s.e.m. *P<0.05, Student's paired t-test. Scale bars: 50 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 6. Pds5B-/- MEFs lack sister chromatid cohesion defects. (A,B) Metaphases from mouse embryonic fibroblasts (MEFs) derived from WT and Pds5B-/- mice show no differences in sister chromatid cohesion. (C) Multi-species alignment of AT hook domains of PDS5B. Hs, Homo sapiens; Pa, Pan troglodytes; Ca, Canis familiaris; Mm, Mus musculus; Rn, Rattus norvegicus; Ga, Gallus gallus; Xe, Xenopus tropicalis; Da, Danio rerio; Te, Tetraodon nigroviridis. (D) Subcellular localization of human homologs of PDS5 using EGFP-tagged PDS5A and PDS5B fusion proteins. HeLa cells were transfected with EGFP-PDS5A and EGFP-PDS5B using Lipofectamine. At 24 hours later, the EGFP signals were examined. Left panels are phase-contrast images of HeLa cells; right panels are images of GFP signals. Note that EGFP-PDS5B is present in the nucleus and highly enriched in the nucleolus (arrows), whereas EGFP-PDS5A is localized to the nucleus but excluded from the nucleolus (arrows). Scale bar: 5 µm.

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