Article Figures & Data
Figures
- Figure 1.
Targeted inactivation of Ddo. A, Ddo−/− mice were created by targeted removal of exon 3 via homologous recombination with a neomycin resistance gene (neo), introducing a frame shift. Southern blot was performed with the 5′ probe of BamHI-digested genomic DNA (12.8 kb band in the Ddo+ allele; 4.3 kb band in the Ddo− allele). tk, Thymidine kinase. B, Northern blot of total RNA with a Ddo exon 2–4 probe. The arrowhead denotes potential truncated Ddo transcript. C, Western blot of P2 fraction lysate. Cyto. C, Cytochrome c.
- Figure 2.
Ddo−/− mice display elevated d-aspartate levels. A, HPLC amino acid analysis of 4-week-old kidney d-aspartate (Ddo+/+ vs Ddo+/−, p = 0.021; Ddo+/− vs Ddo−/−, p = 0.008). B, Four-month-old kidney d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.004). C, Four-month-old brain d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.0009). D, Four-month-old adrenal d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.00001). E, Four-month-old ovarian d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.000004). F, Four-month-old testicular d-aspartate (Ddo+/+ vs Ddo−/−; p = 0.0001) and l-aspartate (Ddo+/+ vs Ddo−/−; p = 0.013). *p < 0.05; **p < 0.01; ***p < 0.001; comparison with two-tailed Student’s t test; data are expressed as mean ± SEM.
- Figure 3.
Elevated d-aspartate in Ddo−/− mice pituitary intermediate lobe is associated with diminished melanocortin levels. A, Ddo in situ hybridization (n = 2). B, d-Aspartate immunohistochemistry (n = 4). C, D, POMC in situ hybridization in the pituitary (C; n = 3) and arcuate nucleus (D; n = 2). E, POMC Western blot of crude pituitary lysate (n = 2). F,G, Crude pituitary lysate dot blot (F; n = 4) and immunofluorescence (G; n = 2) of α-MSH. Scale bars, 100 μm. a, Anterior lobe; i, intermediate lobe; p, posterior lobe.
- Figure 4.
Ddo−/− mice display diminished melanocortin-dependent behaviors. A, Body mass measured at indicated ages (2 months, p = 0.03; 7 months, p = 0.007; 12 months, p = 0.004). B, Latency of male mouse to mount (p = 0.01) or intromit (p = 0.025) estrous CD-1 female. C, D, Time spent autogrooming during resident–intruder aggression test (C; n = 8 pairs; p = 0.04) and elevated plus maze test (D; p = 0.02). *p < 0.05; **p < 0.01; comparison by two-tailed t test; data are expressed as mean ± SEM. Sig., Significance.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplemental Figure 1. Juvenile Ddo�/� mice display elevated D-aspartate levels A, HPLC amino acid analysis of 4-week brain D-aspartate Ddo+/+ vs. Ddo�/�; p = 0.0004. B, 4-week adrenal D-aspartate Ddo+/+ vs. Ddo�/�; p = 0.005, and C, 4-week testicular D-aspartate Ddo+/+ vs. Ddo�/�; p = 0.0001 and L-aspartate Ddo+/+ vs. Ddo�/�; p = 0.015. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; comparison using two-tailed t-test; data are expressed as mean � SEM).
- supplemental material - Supplemental Figure 2. D-Aspartate immunohistochemistry in Ddo�/� and wild-type brain. D-Aspartate immunohistochemistry in wild-type mice resembles rat findings with light immunoreactivity in cerebral cortical neurons (A, arrow), neurons in the hilum of the dentate gyrus (B, arrow), hippocampal CA3/CA2 neurons (C), hypothalamic paraventricular nucleus (D), hypothalamic supraoptic nucleus (E), and pineal gland (F). D-Aspartate is not observed in CA1 neurons in wild-type or Ddo�/� hippocampus (C). Additionally, central nervous system cells normally containing D-aspartate are more strongly stained in the Ddo�/� mice as seen in cerebral cortical neurons (A; arrowhead), neurons in the hilum of the dentate gyrus (B, arrowhead), hippocampal CA3/CA2 neurons (C), and pineal gland (F). Each experiment was performed two independent times at 4 weeks and at 4 months with similar results. (Scale bars = 100 μm; 3V = 3rd ventricle; O.N. = optic nerve; DG = dentate gyrus; CA = cornu ammonis).
- supplemental material - Supplemental Figure 3. D-Aspartate immunohistochemistry in peripheral organs of Ddo�/� and wild-type mice. Wild-type kidney is normally devoid of D-aspartate; however, D-aspartate appears in both Ddo�/� mouse kidney cortex and medulla (A & B). Ddo�/� renal cortical staining is restricted to Bowman�s capsule (A, arrow). Ddo�/� renal medullary staining is found in the thin limbs of the Loops of Henle (B, arrow) as evidenced by immunopositive cells having a thin epithelia, nuclei bulging into the cytoplasm, and round shape on transverse section (data not shown). Unlike rat, mouse D-aspartate is not observed in the 4-week wild-type adrenal medulla (C) but is present in epinephrine-producing chromaffin cells of the Ddo�/� adrenal medulla (C, asterisk). Similar to the rat, wild-type D-aspartate in mice localizes to the innermost rim of the seminiferous tubules suggestive of elongate spermatids (D, asterisk). Ddo�/� testes display this pattern (D, double asterisk) and show new immunoreactivity in Leydig cells (D, arrow). Each experiment was done two independent times at 4 weeks and 4 months yielding similar results. (Scale bars = 100 �m).
