Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse

Behavior of Dync1li1^N235Y/N235Y mice. A, Dync1li1^N235Y/N235 mice displayed significantly lower levels of spontaneous locomotor activity compared with wild-type littermates. B, On the elevated plus maze (EPM), the Dync1li1^N235Y/N235Y mice spent significantly less time in the open arms and more time in the closed arms. C, They also made significantly fewer entries to both the open and closed arms. D, On the successive alleys task Dync1li1^N235Y/N235Y mice spent less time in the more anxiogenic alleys compared with wild types. E, They also made fewer entries to these alleys (the Dync1li1^N235Y/N235Y mice did not make any entries to alleys 3 or 4). F, Footprint analysis using the CatWalk system found that compared with wild types, Dync1li1^N235Y/N235Y mice displayed a significantly greater difference between the BOS of their front legs and hind legs due to a relative narrowing of the front BOS and a widening of the hind BOS; a representative sample from one animal is shown. Green, Right front foot; dark green, right hind foot; red, left front foot; dark red, left hind foot. Animals walked from right to left.

Morphology of wild-type and mutant Dync1li1^N235Y/N235Y cortical neurons. A, Cortical neurons were electroporated in vivo at E15, then cultured in vitro from E17 embryos. At 10 DIV they were analyzed to compare morphology of wild-type and Dync1li1^N235Y/N235Y cortical neurons. GFP-positive neurons (i) were hand-traced using Neurolucida software (ii). B, The length of dendritic trees for wild-type and Dync1li1^N235Y/N235Y neurons, in vitro. i, Dync1li1^N235Y/N235Y dendrites were significantly longer than in wild-type neurons (p = 0.0029). ii, Dync1li1^N235Y/N235Y dendrites terminated significantly further from the soma than wild-type dendrites (p = 0.00281). C, Low magnification of the cortex from a wild-type brain electroporated with GFP at E15 and harvested at P15. GFP-positive neurons occupy a discreet band corresponding to cortical layers II/III. D, i, Higher-magnification image showing the orientation of a pyramidal neuron in layer II. ii, Neighboring neurons align in the same orientation with apical dendrites projecting toward the pial surface. E, The cortex from a Dync1li1^N235Y/N235Y brain electroporated with GFP at E15 and harvested at P15 (rostrocaudally matched with C). Compared with C, GFP-positive cells were more distributed, with more neurons residing in the uppermost part of the cortex (arrows). F, The cell body and apical dendrite of neurons located closer to the pial surface in Dync1li1^N235Y/N235Y mutants were mis-oriented. G, Neurons in layer II of Dync1li1^N235Y/N235Y cortex also often displayed incorrect orientation. H, Comparison of the proportion of electroporated neurons located in upper layers of the neocortex, as measured by fluorescence intensity. I, Higher-magnification imaging from wild-type cortex in more caudal sections showed most GFP-positive neurons within layer II/II. J, In contrast to I, GFP-expressing cells in Dync1li1^N235Y/N235Y cortex occupied a location much closer to the pial surface. K, The apical dendrite of neurons Dync1li1^N235Y/N235Y cortex were often thickened and projected in an incorrect orientation. Scale bars: A, C, E, I, J, 100 μm; D, F, G, K, 50 μm.

Dync1li1^N235Y point mutation increases branching in cultured sensory neurons. A, Fluorescent images of DRG neurons from wild-type and Dync1li1^N235Y homozygous mice under naive and injury conditions. Right leg sciatic nerves of wild-type and mutant mice were crush-lesioned 3 d before dissection of L4–5 DRGs for primary culture of sensory neurons; corresponding contralateral DRGs were used for naive controls. Cultures were imaged by automated fluorescent microscopy after 20 h in vitro, to obtain 150 images per slide, at magnification 10×. B, Quantification shows a significant increase in branching of naive sensory neurons from Dync1li1^N235Y/N235Y homozygotes (29.4% increase over wild type). Increased branching was also observed in homozygous Dync1li1^N235Y/N235 sensory neurons after injury, although the difference is less pronounced than in naive neurons, apparently due to a more robust injury response in the wild-type neurons. ***p < 10⁻⁶; *p < 0.05 (Student's t test). Scale bar, 100 μm.

Neurophysiological and histological analysis of Dync1li1^N235Y/N235Y mice. A, Dync1li1^N235Y/N235 have mice a significant reduction of the peak-to-peak amplitude of A-fiber compound action potential in the saphenous nerve compared with wild-type littermates. *p < 0.05. B, QSum plots of the neuronal profile area distribution of neurofilament-immunoreactive neurons (giving rise to large myelinated A-fibers; round symbols) and of peripherin-immunoreactive cells (giving rise to C-fibers; square symbols) in lumbar dorsal root ganglia. The cell profile size in both populations is reduced in Dync1li1^N235Y/N235Y mice compared with wild-type littermates.

Golgi reassembly in wild-type and Dync1li1^N235Y/N235Y MEFs. A, MEFs were treated with cold (4°C) for 20 min and nocodazole for 3 h, washed and then allowed to recover at 37°C for the times indicated. Golgi are shown in red, cytoskeleton in green (α-tubulin) and nuclei in blue (DAPI). The scale bar represents 30 μm. B, The total number of discrete spots and the total area of Golgi complex were measured to calculate the ratio of spots/total area per cell; 5 cells per genotype were assessed. In wild-type and heterozygous cells, after 50 min of recovery, the Golgi complex has reformed and is not significantly different from that in untreated cells. However, in the Dync1li1^N235Y/N235Y homozygous mutant cells, the Golgi complex has a significant defect in recovery after 50 min compared with untreated cells and treated wild-type and heterozygous cells at the same time point.