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doi: 10.1242/10.1242/dev.00498

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Notch activity in neural cells triggered by a mutant allele with altered glycosylation

¹ Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
² Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, The State University, Piscataway, NJ 08854, USA

View larger version (89K): [in a new window]   Fig. 1. Mosaic analysis with Dl. (A) Eye imaginal disc (anterior towards the left). Dl protein (magenta) accumulates in the differentiating region posterior to the morphogenetic furrow (arrowhead). Mitotic cells (labeling for phosphorylated histone H3 in green) are absent from the morphogenetic furrow region where the cell cycle is arrested. R8 cells are always specified from cells born anterior to the morphogenetic furrow when Dl protein is undetectable. (B) Phase-contrast micrograph of an eye section through the apical R7 level. Photoreceptor cells homozygous mutant for Dl are detected by absence of dark pigment granules at the rhabdomere base. Two ommatidia in this section lack any Dl mutant photoreceptor cells (green stars). All the other ommatidia contain one or more mutant cells. A variety of developmental defects are seen in these ommatidia. (C) Bright-field micrograph through the same eye at the basal R8 level. Every R8 cell is pigmented and therefore wild type for Dl (arrows).

View larger version (110K): [in a new window]   Fig. 2. Dl overexpression. (A,C) Adult eyes (anterior towards the left). (B,D) Details of eye imaginal discs labeled for the R8-specific protein Boss. (A,B) The wild-type pattern of ommatidia (A) and R8 cells (B). (C,D) Similar numbers of ommatidia and R8 cells in the GMR>Dl genotype (the adult eye is abnormal because of developmental defects that occur after R8 specification).

View larger version (126K): [in a new window]   Fig. 3. Defective development in spl mutants. (A) Wild-type eye disc labeled for Senseless protein (magenta) and apoptotic cells with activated caspases (green). Posterior to column 0 in the morphogenetic furrow, Senseless labels one R8 cell in each ommatidium. Anterior to column 0, Senseless is expressed in a single column of 'intermediate groups', clusters of six to ten cells from which each R8 cell emerges. (B) spl mutant eye disc labeled as in A. Anterior to column 0, the intermediate groups contain fewer cells often expressing Senseless at lower levels than wild type. Posterior to column 0 many R8 cells are missing and those remaining express Senseless at varying levels. In the posterior of the eye disc cell death is elevated compared with wild type. (C) Detail from spl; GMRp35 eye disc. Cell death is prevented but caspase activation still occurs. Arrowhead indicates caspase activation in an R8 cell. R8 expression of Senseless is variable despite the blockade to cell death. (D) Wild-type eye disc labeled for the photoreceptor marker ELAV (green; left and middle) and R8-specific marker Boss (magenta; left and right). Note the consistent level of Boss expression. (E) spl; GMRp35 labeled as in D. Note the inconsistent Boss expression levels and variable numbers of photoreceptor cells. Arrow indicates a one-cell ommatidium lacking any Boss-expressing R8 cell.

View larger version (97K): [in a new window]   Fig. 4. Targeted activation of N in R8. Arrows indicate missing ommatidia or ommatidia with too few photoreceptor cells. (A) Wild-type pattern of R8 cells labeled for Boss (top) and all photoreceptors labeled for ELAV (bottom). (B) N intracellular domain targeted to R8 with the 109-68 Gal4 line eliminates most R8 cells and other photoreceptor cells also (NEB37D). (C) Boss labeling of the spl mutant shows defects in R8 patterning (top). Elav labeling (bottom) shows both missing and incomplete ommatidia (arrows). (D) R8 and other defects comparable to spl seen when 109-68 targets R8 expression of N intracellular domain from a more weakly-expressing transgene insertion (NEB5A). (E) R8 and other defects comparable to spl seen when 109-68 targets R8 expression of full-length N. (F) R8 and other defects comparable to spl seen when 109-68 targets R8 expression of the N target gene E (spl)m.

View larger version (110K): [in a new window]   Fig. 5. R8 cells are sensitive to Dl in the spl mutant. (A,B) Boss labeling of R8 cells in spl mutant eye discs. (C,D) SEMs of adult eyes from spl mutants. (B,D) R8 cell number, adult eye size and number of ommatidia are significantly reduced by elevated Dl expression targeted posterior to the morphogenetic furrow by GMR Gal4. Compare with the insensitivity of R8 cells to targeted Dl expression in the absence of the spl mutation (Fig. 2).

View larger version (148K): [in a new window]   Fig. 6. Some Notch functions are unaffected by spl. (A) Clone of spl mutant cells revealed by absence of ß-galactosidase (magenta; left and right). Atonal expression (green; left and middle) initiates similarly in spl mutant and wild-type territories. Maintenance of Atonal in R8 cells is defective in spl mutant territories (arrow). (B) Clone of spl mutant cells revealed by absence of ß-galactosidase (magenta; left and right). Senseless expression (green; left and right) initiates similarly in spl mutant and wild-type territories. Maintenance of Senseless in R8 cells is defective in spl mutant territories (arrows). (C) m0.5-lacZ reporter expression predominantly in R4 cells of wild-type retina. (D) m0.5-lacZ reporter expression only in R4 cells of spl mutant retina. Expression is absent from some ommatidia where R4 cells may be affected. (E) Sections through spl mutant eyes reveal ommatidia containing multiple cells of R7-like morphology (arrow). This aspect of the spl phenotype is not cell autonomous in mosaics (not shown).

View larger version (76K): [in a new window]   Fig. 7. Fucosylation of EGF repeats from wild-type and Fringe. (A) Proteins containing EGF repeats. Protein EGF13-15 includes EGF repeats 13-15 from wild type N, flanked by V5 and 6His tags. There is a potential O-fucosylation site in EGF repeat 13 (T540; blue T). Protein EGF13-15 I578T carries the Ile578 to Thr578 substitution (red T) from the spl allele. There are potential O-fucosylation sites in EGF repeat 13 and 14. Protein EGF13-15T540I resembles protein EGF13-15 with Thr540 substituted by Ile. This protein has no potential O-fucosylation site. Protein EGF13-15T540I,I578T is same as protein EGF13-15I578T, except that Thr540 is substituted into Ile. Ile578 is the only potential fucosylation site. (B) Autoradiographs of radiolabeled proteins after membrane transfer. EGF13-15T540I protein is not labeled (lane 3), unlike the other proteins. (C) Immunoblotting with anti-His6 control for equal protein loading. Red asterisk indicates mutation.

View larger version (70K): [in a new window]   Fig. 8. The spl mutation affects R8 independent of Fringe. Clones of fng homozygous mutant cells were identified by a lack of ß-galactosidase expression (magenta; A,A'',B,B''). R8 cells were labeled for Senseless protein (green; A,A',B,B'). (A) Senseless expression was largely normal in the absence of fng. (B) The spl mutation reduced the number of R8 cells in fng mutant cells and in neighboring territories. As was typical in the spl mutant, the level of Senseless expression varied in cells that were also mutant for fng (arrows).