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doi: 10.1242/10.1242/dev.00158

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Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster

¹ Zoology Department, Stockholm University, S-106 91 Stockholm, Sweden
² Institute of Genetics, University of Mainz, Saarstrasse 21, D-55122 Mainz, Germany
³ European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
⁴ Center of Molecular Biology Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

View larger version (151K): [in a new window]   Fig. 1. Ultrastructural comparison of the central nervous system in transheterozygous Df(2L)32FP-5;sal445 mutant (A,B,E,F) and wild-type (C,D,G,H) embryos at early stage 16 (left panel) or 17 (right panel) of embryonic development. In the mutant CNS at early stage 16 (A), cell bodies are separated by large extracellular spaces occupied by vacuoles and other membranous material (arrows). At this stage neuronal cell bodies in wild-type tissue are tightly packed and the very thin extracellular space, free from vacuoles or other membranous material, is sharply outlined by the smooth cell membranes of CNS cells (C). The phenotype is not longer expressed a few hours later, when the extracellular space in the mutant CNS (B) has the same size as in wild-type tissue (D) and is mostly free from the membranous material found earlier. In the mutant neuropil at stage 16 (E), large vacuoles (arrows) separate `clumps' of axons and filopodia, in contrast to the wild-type neuropil (G), where axons and filopodia are separated by a `clean' extracellular space. The insets in E and G show representative axonal profiles at higher magnification. Notice the smaller diameter of axons in sal mutant CNS. By stage 17, neuronal processes in the neuropil are equally tightly packed in both mutant (F) and wild-type tissue (H) and most of the extracellular vacuoles previously found in the mutant have disappeared. All sections depicted here are from thoracic neuromeres of the nerve cord but the same morphology was observed across different regions of brain and nerve cord. Scale bar: 2µm.

View larger version (82K): [in a new window]   Fig. 2. Expression of cell adhesion markers in sal mutants. (A) Confocal optical sections show representative views of the expression of cell adhesion proteins in anterior nerve cord of embryos heterozygous (top row) or homozygous (bottom row) for the sal mutation at the stage when the strong adhesion phenotype was detected with TEM (early stage 16). (B) Fluorescence levels were consistently lower for Armadillo, N-Cadherin, Neuroglian, Fasciclin 2 and Fasciclin 3, but higher for Notch and normal for Neurotactin. Notice that Armadillo, N-Cadherin, Neurotactin and other markers are expressed by all CNS cells. The bars represent average levels for heterozygotes (black) and sal nulls (grey). Each bar shows the mean value for a minimum of five embryos processed, scanned and measured as detailed in Materials and Methods. Standard error is indicated. The most significant differences were found for Fasciclin 2 (P=0.001), Fasciclin 3 and N-Cadherin (P<0.005) and Armadillo (P<0.05). (C) When wild-type embryos were included in the comparison, the fluorescence levels for Armadillo, and N-Cadherin showed a gradual discrepancy from wild-type to null mutant embryos. The example shown here is Armadillo. Scale bar: 10 µm.

View larger version (17K): [in a new window]   Fig. 3. Dynamic expression of adhesion proteins in the central nervous system. In wild-type embryos, the fluorescence levels measured by laser confocal microscopy for Armadillo, Fasciclin 2 and Neuroglian changed significantly within a short developmental interval. Each bar represents the mean value for a sample of at least six embryos and the s.e.m. is indicated. An increase was found for Armadillo (P=0.005), Fasciclin 2 (P=0.003) and Neuroglian (P=0.001); significance was calculated with the Student's t test. See Materials and Methods for the conditions used for immunostaining, confocal microscopy and quantification of fluorescence levels.

View larger version (93K): [in a new window]   Fig. 4. In vitro analysis of neuronal clones derived from single CNS precursors taken from sal null (A,C,E) or wild type (B,D) embryos. After 18 hours of culture, mutant clones (A) had thinner and poorly branched fibres compared with wild-type clones (B). After 4 days in culture, the branches in mutant clones (C) were still less elaborated than in wild-type clones (D). Mutant clones were often surrounded by cell debris (C) and their branches formed `anastomosing' structures (arrows in E) not observed in wild-type clones. Staining for the glial marker Repo (arrowhead in E) indicated that mutant clones comprised neuronal and glial cells.

View larger version (76K): [in a new window]   Fig. 5. Time-lapse video analysis of cell cultures derived from sal nulls single neuronal precursors. Abnormal networks were formed by mutant fibres in which individual anastomoses were formed and disassembled over short time periods (arrows in A-D). Another abnormal feature was the formation of vacuolar-like `blisterings' along the axons that persisted for short periods before becoming reabsorbed (arrow in E-H).

View larger version (55K): [in a new window]   Fig. 6. Abnormalities in the cytoskeleton of sal mutant neurones in vitro (A-C) and in situ (D-E). Tubulin staining in vitro uncovered irregularities in the distribution of tubulin along the axon. The staining did not reach the growth cone (arrow in A) and had sometimes interruptions along the axon (arrows in B). Compare with the wild-type neurones in C [double stained with anti-tubulin (in red) and anti-HRP (in green)], where tubulin staining is uniformly intense along the axons and reaches almost the distal border of the growth cone (arrow). (D) At the stage of the strong TEM phenotype (early stage 16) laser confocal microscopy showed normal spatial distribution of F-actin, tubulin, and the microtubule associated protein Futsch/22C10 in sal mutant CNS. Notice the wild-type pattern in both heterozygotes and homozygotes, with accumulation of the marker along nerve roots and major axonal tracts. Careful analysis at higher magnification revealed frequent `mild' malformations in the organisation of axonal tracts in sal null condition (not shown). (E) When the fluorescence levels were measured (see Materials and Methods) the homozygotes were found to express higher levels for Actin, and lower for Tubulin (P<0.05) and Futsch (P=0.05). Each bar represents the mean values for samples of at least six embryos each and the s.e.m. is indicated.

View larger version (98K): [in a new window]   Fig. 7. sal mutant cell lineages (at stage 16) derived from single neuroectodermal precursors in mutant (A-E) or wild-type (F-I) background. Clones in A-E were obtained by labelling precursor cells (at stage 7) with DiI in situ, clones in F-I were obtained from HRP-labelled precursors upon transplantation (at stage 7) from mutant donors into wild-type hosts. All clones are located within abdominal neuromeres (A1-A4, shown as horizontal views, anterior towards the left). Drawings with light background (in A,B,D,F,G,I) show camera lucida tracings of the respective preparations. Drawings with dark background (in B-D,G-I) show identified wild-type lineages for comparison (see Bossing and Technau, 1994; Bossing et al., 1996; Schmidt et al., 1997). Glial cells are shown in green, neuronal cell bodies are in red and fibre projections are in black (see also arrows in B,C). In both series of experiments, there was a wide range of clonal phenotypes, including cases with no similarities to wild-type clones (A,F), cases with some components showing similarities to wild-type clones (B, NB1-3; G, NB4-2) and cases very similar to wild type clones (C, NB7-4; H, NB3-3). Along axons spherical thickenings were found (E, arrows). Although sal is not expressed in the CNS midline, some midline clones showed irregularities in their projection patterns (D, VUM clone; I, UMI clone).