Amino acid sequence and molecular domain structure of Dasm1. (A) Dasm1 encodes a 1,178-aa protein with an extracellular domain containing five Ig repeats and two fibronectin III repeats, a single transmembrane (TM) domain, and a large cytoplasmic domain with a type I PDZ domain binding motif at the end. Single-letter abbreviations for amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. (B) An illustration of molecular domain structure of Dasm1 and its similarity to human KIAA1355, Drosophila Turtle, and human Roundabout 1 (H-Robo 1). The positions of the Ig, fibronectin III, and TM domains and the percentages of amino acid identity/similarity between Dasm1 and KIAA1355, Turtle, and H-Robo 1 are shown.

Expression of Dasm1 in the mouse brain. (A) In situ hybridization analysis of Dasm1 expression. Embryonic day 18 mouse embryo sections were probed with digoxigenin-labeled antisense (Left) and sense (Right) sequences of Dasm1. Note the high expression of Dasm1 in the brain, especially in the forebrain (neopallial cortex, NC), intermediate zone, and ventricular zone. CP, cerebellar primordium; MB, midbrain; MO, medulla oblongata; Th, thalamus. (B) Western blot analysis of Dasm1 expression in the brain. Crude membrane protein extracts from adult rat brains were immunoblotted with the polyclonal antiserum against the C terminus of Dasm1 (Right) and the preimmune serum (Left). The anti-Dasm1 serum strongly recognizes a band ≈130 kDa but not the preimmune serum (arrow). Molecular mass markers are listed on the left. (C) Adult mouse brain sections (≈7 μm) were stained with the polyclonal antiserum against Dasm1 (green, Top) and, propidium iodide (PI), a dye, labels the DNA (red, Middle). The overlay is shown in Bottom. Antibody against Dasm1 (green) showed punctate staining in both the cell bodies and dendrites (Left) of hippocampal and cortical neurons, whereas the preimmune serum gave no detectable staining (Right). High-magnification images of the staining in cell body (a) and apical dendrites (b) of hippocampal CA1 pyramidal neurons (squares in Left) are shown. [Scale bars (from left to right) = 250, 20, 20, and 250 μm.] (D) Immunohistochemistry analysis of Dasm1 in the cerebellum. ML, molecular layer; WM, white matter. (Scale bar = 500 μm.)

Expression of Dasm1 in developing dendrites. Expression of Dasm1 in the dendrites of developing neurons is shown. Hippocampal dissociated neurons cultured for 4-5 days in vitro (DIV) were double-labeled with antibodies against Dasm1 (green) and MAP2 (red), a protein marker for the dendrites. Note that Dasm1 was expressed in the dendrites of these neurons, which undergo dynamic outgrowth at this stage. (Scale bar = 25 μm.)

Efficacy and specificity of Dasm1 RNAi. (A and B) Western analysis of Dasm1 RNAi plasmids on Dasm1 and Robo1 expression in Cos-7 cells. Compared with the control RNAi against a randomly scrambled sequence, Dasm1 RNAi b and c showed a suppression in Dasm1 expression, whereas Dasm1 RNAi a did not show an obvious effect on Dasm1 expression (A). In contrast, none of Dasm1 RNAi plasmids showed obvious effect on hemagglutinin-Robo1 expression compared with control RNAi (B). An equal amount of protein (≈25 μg) was loaded for each lane, and the same sample was blotted against α-tubulin antibody. (C) Quantification of Dasm1 RNAi plasmids on Dasm1 and Robo1 protein expression in Cos-7 cells.

Suppression of Dasm1 expression impairs dendrite outgrowth. (A and B) Dasm1 RNAi had no obvious effect on axonal arborization. Neurons transfected with either Dasm1 RNAi plasmid (B) or control RNAi plasmid (A) together with EGFP were stained with the dendritic marker MAP2 antibody. Extensive EGFP fluorescence-labeled axonal arbors lacking MAP2 staining (indicated with arrows and inside dotted lines) were seen in both groups. (Scale bar = 50 μm.) (C and D) Neurons transfected with Dasm1 RNAi/EGFP (D) showed defects in dendrite development, compared with those transfected with control RNAi/EGFP (C). Neurons were double-labeled with antibodies against MAP2 to visualize dendritic arbor (red) and Dasm1 to monitor the expression level of endogenous Dasm1 (blue) after RNAi treatments. The EGFP channel is shown in Left and the overlay is shown in Right. Arrows indicate the axon and arrowheads indicate the positions of cell body of transfected neurons. (Scale bar = 50 μm.) (E) Correlation between the expression level of Dasm1 and the complexity of dendritic arbor. The dendritic branch lengths of neurons transfected with EGFP/Dasm1 RNAi (red circle) or EGFP/control RNAi (black circle) were plotted against the Dasm1 staining intensity at the cell bodies. Note that there was a significant reduction in average Dasm1 staining intensity at the cell bodies with Dasm1 RNAi when compared with the control RNAi (P < 0.05; open circles). AU, arbitrary unit. (F) The average total dendritic, but not axonal, branch length was significantly reduced in neurons transfected with Dasm1 RNAi compared with that of the control neurons (^**, P < 0.001).

Dasm1 lacking the intracellular tail acts as a dominant negative and impairs dendrite outgrowth. (A and B) Neurons expressing Dasm1(delC)-EGFP (B) showed defects in dendrite development when compared with control neurons expressing EGFP alone (A). The dendritic arbor was visualized with MAP2 antibody staining (Center). The EGFP channel is shown in Left, and the overlay is shown in Right. (Scale bar = 50 μm.) (C) The average total dendritic branch length was decreased in neurons expressing Dasm1(delC)-EGFP or enhanced cyan fluorescent protein (ECFP)/Dasm1(delC)-EGFP compared with control neurons expressing EGFP or ECFP alone (^**, P < 0.001). (D) Sholl analysis showed that the dendritic arbor was severely reduced in neurons expressing Dasm1(delC)-EGFP (n = 22) compared with control neurons expressing EGFP alone (n = 21). Concentric circles with a 20-μm spacing were drawn around the cell body, and the number of intersections of all dendritic branches with the circles was counted.