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First published online 10 January 2007
doi: 10.1242/dev.02758

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Distinct functions of -Spectrin and ß-Spectrin during axonal pathfinding

Jörn Hülsmeier¹, Jan Pielage^1,*, Christof Rickert², Gerd M. Technau², Christian Klämbt^1, and Tobias Stork¹

¹ Institut für Neurobiologie, Badestr. 9, 48149 Münster, Germany.
² Institut für Genetik, Saarstr. 21, 64123 Mainz, Germany.

View larger version (153K): [in this window] [in a new window]   Fig. 1. The karussell phenotype. Dorsal view of dissected embryonic nervous systems stained for BP102 expression. Anterior is to the left. (A) In wild-type stage-14 embryos, commissures are established as regular axon fascicles. (B) In wild-type stage-16 embryos, the typical ladder-like axon pattern is established. Two clearly separated commissures (anterior commissure, ac; posterior commissure, pc) are found in every neuromere. Neuromeres are connected by the longitudinal connectives (lc). (C) In stage-14 hemizygous-mutant ß-spectrinkusS012 embryos, characteristic loops are found at the point where commissural axons normally enter the connectives (arrows). (D) Stage-16 hemizygous ß-spectrinkusS012 embryo. The longitudinal connectives are found closer to the midline and appear thinner (arrowhead); in addition, the segmental commissures are not clearly separated and appear slightly fused (arrow). (E,F) Hemizygous ß-spectrinkusS012 embryos that express ß-spectrin under the control of the elav promoter. The axonal phenotype is rescued.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Molecular characterization of ß-spectrin mutants. (A) Schematic drawing of the 2291-amino acid large ß-Spectrin protein. The position of the S012 mutation is indicated. The bar indicates the part of the ß-Spectrin protein used for immunization. (B) Western blot of cell extracts derived from stage 15-17 embryos probed with a polyclonal antiserum generated against a ß-Spectrin fusion protein. First lane: wild type (wt), the 280 kDa large ß-Spectrin protein and a 200 kDa degradation product is visible. The 50 kDa band is a background band recognized by the polyclonal antibody. Mutant ß-spectrin embryos were selected using GFP-carrying balancer chromosomes. The genotype is indicated. In em6, em15, em21, G113, E292, L105 and M046 hemizygous embryos, truncated ß-Spectrin proteins are detected. E175, H127 and S012 hemizygous-mutant embryos lack detectable ß-Spectrin expression. (C) The same membrane was probed for expression of -Spectrin. Notice the strong reduction of -Spectrin expression in all genotypes, regardless of whether residual ß-Spectrin protein can still be detected or not. (D) Coomassie staining of same membrane to demonstrate equal loading. ABD, actin-binding domain; SR, spectrin repeats; Ank, ankykrin domain; PH, pleckstrin homology domain.

View larger version (104K): [in this window] [in a new window]   Fig. 3. Expression of ß-Spectrin in the nervous system. Confocal views of dissected nervous systems of wild-type stage-16 embryos stained for expression of ß-Spectrin (white), axonal membranes (anti-HRP, green) or the SemaIIbMyc marker (red). (A) In wild-type embryos, ß-Spectrin expression is found in neuronal cell bodies and specific axonal fascicles (arrowheads). The arrow denotes increased levels of ß-Spectrin at the CNS midline. (B) In homozygous-mutant gcm embryos, lateral glial cells are absent. ß-Spectrin can still be detected at specific fascicles (arrowheads) and at the CNS midline (arrow). (C,C') The SemaIIbMyc marker is expressed by only one neuron per hemineuromere. This neuron is positioned at the lateral margin of the neuropil. SemaIIbMyc-positive axons project across the midline in the anterior commissure and then make a sharp turn to follow a specific path in the longitudinal connective. (D,D') In hemizygous-mutant kusS012 embryos, the SemaIIbMyc-positive neurons appear normally specified, but show irregular positions in the nerve cord, often being displaced towards the CNS midline. The SemaIIbMyc-positive fascicles are found closer to the CNS midline (double-headed arrow indicating distance in C and D) and the fascicle morphology appears changed. Often, ectopic projections are found that may correspond to enlarged growth cones (arrows). In addition, the precision in axonal pathfinding is lost; however, we did not observe ectopic crosses of the CNS midline.

View larger version (110K): [in this window] [in a new window]   Fig. 4. Non-autonomous effects of ß-spectrin on axonal patterning. Dissected preparations of stage-16 embryos stained for HRP (green) and ß-Galactosidase or ß-Spectrin, as indicated. (A,A') The ap::Gal4 driver directs lacZ expression into a small subset of ipsilateral-projecting neurons. (A') Notice the distance between the ß-Galactosidase-expressing fascicles (yellow bar). (B,B') Following expression of ß-spectrin in the ap::Gal4 pattern in kus mutants, the axonal trajectories are found at the same distance from the CNS midline as in hemizygous-mutant kus-mutant embryos (C,C'). (D,D') The sim::Gal4 driver directs lacZ expression into the MP1 neurons and some midline cells. Notice the distance of MP1 fascicles to the midline (yellow bar). (E,E') Following expression of ß-spectrin in the sim::Gal4 pattern in kus mutants, the MP1 axons are found at the same distance from the CNS midline as in hemizygous kus-mutant embryos (F,F').

View larger version (98K): [in this window] [in a new window]   Fig. 5. Morphology of the karussell-mutant neurons. Individual neuroblasts were filled using the DiI methodology. (A,E) DiI-labeled lineages of neuroblasts NB 3-5 and NB 7-3 in a wild-type embryo. (A') Higher magnification of the dotted area in A. The arrowhead denotes the growth cone. (B,F) Camera lucida drawings of wild-type lineages of NB 3-5 and NB 7-3. (C,G) DiI-labeled lineages of neuroblasts NB 3-5 and NB 7-3 in a hemizygous kus-mutant embryo. (C') Higher magnification of the dotted area in C. The arrowhead denotes the enlarged growth cone in the kus mutant. (D,H) Camera lucida drawings of these preparations reveal principally normal projection patterns, but altered fiber morphology and enlarged growth cones. No ectopic crossing of the CNS midline was observed.

View larger version (90K): [in this window] [in a new window]   Fig. 6. ß-spectrinkus interacts with slit. (A) In wild-type stage-16 embryos, three distinct fascicles can be recognized following Fasciclin II staining. The most medial fascicle never crosses the CNS midline (see C for quantification). (B) In hemizygous-mutant ß-spectrinkus embryos, the inner fascicles appear to cross the midline (arrow). (D) Hemizygous-mutant kus embryo also lacking one copy of slit. The number of ectopic midline crosses increases significantly (arrows). (C) Quantification of the number of midline crosses of Fasciclin II-positive axon bundles. The number of embryos counted, the average number of ectopic crosses per embryo and the standard deviation for each genotype is indicated.

View larger version (126K): [in this window] [in a new window]   Fig. 7. ß-Spectrin regulates -Spectrin stability. Top three panels show eye imaginal discs with homozygous-mutant cell clones lacking ß-Spectrin (red) or -Spectrin (white), as indicated. Single confocal planes are shown. Clones were induced by expressing flp under the eyeless promoter and are marked by either the lack of GFP expression (green) or the loss of Spectrin expression. (A) In cell clones that are homozygous-mutant for the strong hypomorphic allele ß-spectrinG113, significantly reduced levels of ß-spectrin can be detected that appear to localize normally at the cell cortex. Concomitant to the reduction of ß-Spectrin expression we noticed a reduction in the level of -Spectrin. (B) Similar results were obtained for cells homozygous for the ß-spectrinE292 mutation. (C) Clones homozygous for the strong -spectrin allele D4-65 lack -Spectrin protein expression but ß-Spectrin expression is not affected. Almost the entire eye is mutant for -spectrin, except for a small area (arrow). (D) Wing imaginal disc expressing the UAS-ß-spectrin construct under the control of the patched::Gal4 driver. Elevated levels of ß-Spectrin result in a concomitant up-regulation in the level of -Spectrin.

View larger version (55K): [in this window] [in a new window]   Fig. 8. The -spectrin gene is affected in klötzchen mutants. (A-F) CNS preparations of stage-16 embryos stained for BP102 (A-D) or ß-Spectrin (E,F). (A) Wild-type embryos are characterized by a regular axonal pattern with separated segmental commissures and longitudinal connectives. (B) Homozygous E2-26-mutant embryos show partially fused commissures. (C) A homozygous E2-26 mutant with no further background mutations shows no axonal phenotype. (D) When such embryos are allowed to develop at 4°C for 1 day, a fused-commissure phenotype develops. (E) ß-Spectrin protein in the ventral nerve cord of wild-type embryos. (F) In homozygous E2-26 mutants, ß-Spectrin expression in the neuropil appears altered. (G) -Spectrin protein expression in some of the -spectrin mutants. Proteins were isolated from ten stage 16/17 embryos and were separated on a 6% SDS gel. Following western blotting, -Spectrin expression was detected using the monoclonal antibody 3A9. w1118 embryos were used as a wild-type control. If not otherwise indicated, homozygous mutants were used. Genotypes are as indicated. The P-2 mutation does not lead to a detectable truncation of -Spectrin. In N-2 and, possibly, 1.3 mutants, a slight reduction of the -Spectrin protein size is noted. In the deficiency Aprt32, and in the mutants rg41 and E2-26 all zygotic -Spectrin expression is removed. Only maternal -Spectrin expression is visible. (H) To control for equal loading, the same membrane was probed with anti-Kette antibodies.