Journal: The Journal of Neuroscience

Article Title: Phenotypes of Drosophila Brain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

doi: 10.1523/JNEUROSCI.2106-06.2006

Figure Lengend Snippet: A naked runt phenotype of MB γ neurons is unmasked in vitro. A–C, Normal morphology of MBs in female Dmef2-lacZ homozygotes (third chromosome insertion). Projections of sequential optical sections through the MBs of anti-βgal immunostained brains. The scale bar in A applies to A–C. A, w3L, Wandering third instar larva, with normal-appearing medial (med) and dorsal (dor) MB lobes. B, HE + 5 h, Young pupa. Reduced signal intensity in the lobes (arrows) indicates stage-appropriate degeneration of γ neuron axons. cb, Cell body cluster. C, PA, Pharate adult, several hours before adult emergence, with normal-appearing α, β, and γ lobes. The left α lobe is partially obscured by the cell body cluster. D–H, Cultured female pupal MB neurons, isolated at HE + 5 h, cultured for 3 div with media lacking 20E, and visualized by anti-HRP immunofluorescent staining. Arrow, Cell body. The scale bar in D applies to D–F. D, E, Representative βgal-positive (MB γ) neurons homozygous for Dmef2-lacZ (third chromosome). Note the small size and paucity of branches of the neurite arbors. F, Representative γ neuron from a parallel wild-type (201Y UAS-lacZ/+) culture. Note the considerable length and large number of branches. G, Box plots comparing total neurite lengths of Dmef2-lacZ homozygotes (two independent experiments, 1 and 2) with those of 201Y UAS-lacZ/+. The left side shows γ neurons, identified by βgal expression; the right side shows generic brain-neuron neighbors from the same cultures. The number of neurons in each sample is shown in parentheses. βgal-expressing Dmef2-lacZ neurons are greatly reduced in total length compared with control γ neurons (p < 0.0001), whereas there is no significant (n.s.) difference in the length distributions between generic neurons of the two genotypes. H, Polarity Index distributions for the γ neurons shown in G. The control (201Y UAS-lacZ/+) population exhibits the typical wild-type skewed distribution, whereas βgal-expressing Dmef2-lacZ neurons have very reduced polarity.

Article Snippet: To visualize β-galactosidase reporter-gene product, a preabsorbed polyclonal rabbit anti-βgal antiserum (Cappel) was used at 1:5000 and detected with a Cy3-conjugated donkey anti-rabbit antiserum (Jackson ImmunoResearch) at 1:500.

Techniques: In Vitro, Cell Culture, Isolation, Staining, Expressing, Control

Journal: The Journal of Neuroscience

Article Title: Phenotypes of Drosophila Brain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

doi: 10.1523/JNEUROSCI.2106-06.2006

Figure Lengend Snippet: The polarity of MB γ neurons in vitro results from distinct axonal and dendritic arborizations. A, Representative example of Polarity Index distributions of cultured γ neurons and their generic neighbors isolated from the same wild-type pupal brain. Note the skewed distribution of PI values of MB γ neurons, which reflect their highly polar neurite arbors. B, C, Comparison of Nod:βgal and Khc:βgal fusion-protein distributions in subsets of neuronal processes when expressed in γ neurons in vitro. Neurons were isolated from w; 201Y UAS-GFP/+; UAS-Nod:lacZ/+ or w; 201Y UAS-GFP/+; UAS-Khc:lacZ/+ pupae at HE + 5 h. Cells were cultured for 3d and then fixed and double immunostained to detect all neuronal membranes and βgal. B, Comparison of Khc:βgal and Nod:βgal accumulation in the dominant primary processes of γ neurons. Khc:βgal accumulates in all branches, whereas Nod:βgal, which labels the dominant primary process in only 23% of neurons, accumulates primarily in its proximal branches. C, Classification of Nod:βgal and Khc:βgal fusion-protein localization patterns. Every polar neuron from the population sample was classified in one of eight mutually exclusive categories, listed in the center column. Left, UAS-Nod-lacZ driven by 201Y in 138 polar neurons (83% of all labeled neurons in the sample). Right, UAS-Khc-lacZ driven by 201Y in 149 polar neurons (86% of all labeled neurons in the sample).

Article Snippet: To visualize β-galactosidase reporter-gene product, a preabsorbed polyclonal rabbit anti-βgal antiserum (Cappel) was used at 1:5000 and detected with a Cy3-conjugated donkey anti-rabbit antiserum (Jackson ImmunoResearch) at 1:500.

Techniques: In Vitro, Cell Culture, Isolation, Comparison, Labeling

Journal: The Journal of Neuroscience

Article Title: Phenotypes of Drosophila Brain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

doi: 10.1523/JNEUROSCI.2106-06.2006

Figure Lengend Snippet: Nod:βgal and Khc:βgal distribution in MB γ neurons in vitro. Examples of common distribution patterns of the fusion proteins within cultured γ neurons with polar morphologies. Pupal neurons were prepared, cultured for 3 div, and stained as described in Figure 3. Two immunofluorescent images of each neuron are shown: left, negative anti-HRP image, showing neuronal membranes; right, positive anti-βgal image, showing fusion protein accumulation. Arrows indicate the dominant primary process; triangles denote processes with Nod:βgal or Khc:βgal accumulation. The scale bar in A applies to all images. A, B, w; 201Y UAS-GFP/+; UAS-Nod:lacZ/+. A, Nod:βgal in all primary processes except the dominant primary process, the most common pattern of Nod:βgal distribution. B, Nod:βgal in all or most primary processes. but in only the most proximal branches of the dominant primary process. C, D, w; 201Y UAS-GFP/+; UAS-Khc:lacZ/+. C, Khc:βgal in the dominant primary process alone, the most common pattern of Khc:βgal distribution. D, More Khc:βgal in the dominant primary process and less in all others.

Article Snippet: To visualize β-galactosidase reporter-gene product, a preabsorbed polyclonal rabbit anti-βgal antiserum (Cappel) was used at 1:5000 and detected with a Cy3-conjugated donkey anti-rabbit antiserum (Jackson ImmunoResearch) at 1:500.

Techniques: In Vitro, Cell Culture, Staining

Journal: The Journal of Neuroscience

Article Title: Phenotypes of Drosophila Brain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

doi: 10.1523/JNEUROSCI.2106-06.2006

Figure Lengend Snippet: Filagree phenotype identified in brain neurons in primary cell culture. Female brain neurons, harvested from wandering third instar larvae or young pupae (HE + 5 h), cultured 3 div, and visualized by anti-HRP immunostaining. γ neurons were identified by 201Y-driven βgal expression. A, Wild-type pupal γ neuron (201Y UAS-lacZ/+). Note the overall clockwise growth pattern of the largest branches but the spike-like appearance of the terminal neurites. B, C, Representative pupal γ neurons manifesting the filagree phenotype (y npr13 w sn3/Binsn; 201Y UAS-lacZ/+). The phenotype includes striking curvature of nearly all neurites, irregular neurite caliber, and the hook-like appearance of many branch termini. D, Example of filagree jewelry. E, Pupal brain neurons (y npr13 w sn3/Binsn; 201Y UAS-lacZ/+) need not be MB γ neurons to show the filagree phenotype. Anti-HRP (Ei) and anti-βgal (Eii) images of the same field. A single cell is βgal-positive (arrow), but all neurons in the field display the filagree phenotype. F, G, Anonymous larval brain neurons (y npr13 w sn3/Binsn) also manifest the filagree phenotype. H–J, Morphometric comparison of female pupal γ neurons with and without the filagree phenotype (y rbp5 wa sn3/Binsn; 201Y UAS-lacZ/+ and 201Y UAS-lacZ/+, respectively). The number of neurons in each sample is shown in parentheses. H, Box plot distributions of total neurite length (left), territory (center), and branch-point density (right). Both wild-type (wt) control neurons and filagree (fil) neurons respond to 20E with increased total neurite length (p < 0.0001 for each). For a given culture condition, there was no significant difference between the length distributions of wild-type and filagree neurons. However, filagree neurons cover smaller territories (p < 0.0001) and have higher branch-point densities (p < 0.0001) than do wild-type neurons. I, Length-territory relationships. For a given total length, filagree neurons cover less territory than do wild-type neurons. J, Polarity Index distribution of the filagree neurons. In both the presence and absence of 20E, the profiles were very similar to wild-type (Fig. 3A), with the majority of neurons having a highly polar morphology.

Article Snippet: To visualize β-galactosidase reporter-gene product, a preabsorbed polyclonal rabbit anti-βgal antiserum (Cappel) was used at 1:5000 and detected with a Cy3-conjugated donkey anti-rabbit antiserum (Jackson ImmunoResearch) at 1:500.

Techniques: Cell Culture, Immunostaining, Expressing, Comparison, Control