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Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
² Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
³ Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (55K): [in a new window]   Fig. 1. Loss of ventral eye in L mutants. (A,B) Wild-type and Lsi homozygote adult eyes by scanning electron microscopy. (C,D) Wild-type and Lsi third instar eye discs. The morphogenetic furrow and photoreceptor clusters were respectively marked with dpp-lacZ reporter staining (blue) and neural marker Elav (brown). Furrow progression and Elav expression were normal within the remaining eye disc. (E) mirr-lacZ enhancer trap line, mirrB1-12. (F) w–; Lsi; mirrB1-12/+ flies have mostly w+ ommatidia. (G) Wild-type, second instar eye disc stained with anti-ß-gal to label mirr-lacZ+ dorsal cells (brown). (H) In Lsi second instar eye disc, mirr-lacZ+ dorsal domain is considerably larger than the ventral domain (outlined by the broken line). (I) L– mosaic eyes disc generated with Lrev6-3 allele. Clones were visualized by the absence of the lacZ marker (red). Ventral, but not dorsal domain of the eye disc was greatly reduced. A cell junction marker, Dlg (green), was used to outline cells in the disc. (J) Adult mosaic eye showed intact dorsal eye and cuticle-replaced ventral eye. In all figures, white lines mark the DV boundary and broken lines mark the putative ventral domain. Dorsal is upwards and anterior towards the right.

View larger version (75K): [in a new window]   Fig. 2. L mediates N signaling in the ventral eye. (A) Wild-type, third instar disc double-stained with anti-Elav (red) and anti-Dlg (green). (B) UAS-Nintra/dpp-Gal4 caused overgrowth in dorsal and ventral domains (arrows). (C) Lsi mutant showed a loss of the ventral domain. (D) Loss of L suppressed UAS-Nintra/dpp-Gal4 phenotype in the ventral domain. The putative ventral domain is outlined. Dorsal overgrowth persisted, such that the tissue had a crumpled, uneven appearance. (E) UAS-NDN/ey-GAL4 caused a general loss of tissue. (F) Co-expression of UAS-L and UAS-NDN specifically restored the ventral eye. (G) Lsi/+ adult eye showed a characteristic anterior notch (yellow arrow). (H) Removing a copy of N in Lsi/+ animals increased the severity of the L eye phenotype.

View larger version (106K): [in a new window]   Fig. 3. L regulates Ser expression. (A) In wild-type first instar eye disc, Ser was expressed in the ventral domain and at the DV boundary. (B) In Lsi first instar eye disc, ventral Ser expression was lost but posterior midline Ser expression (bracket) persisted. (C) Dlg marked cell boundaries of the eye disc shown in (D-F). (D) Composite of E,F. Ventral Lrev6-3 clone in first instar eye disc showed a decreased Ser expression within the clone (white arrow). However, Lrev6-3 clone near the posterior DV boundary did not affect Ser expression (yellow arrow). There was also a dorsal anterior clone present. (E) Lrev6-3 clones were marked by the absence of lacZ (red). (F) Anti-Ser antibody detected Ser protein expression. (G) Composite of H,I. L flp-out clones (red in H) upregulated Ser expression (green in I) in both dorsal and ventral domains of the first instar eye disc. Dlg staining (blue) marks disc cells.

View larger version (71K): [in a new window]   Fig. 4. Loss of Ser affects ventral growth. (A) Ser– clones, marked by the absence of lacZ (red), did not affect eye size or photoreceptor cluster formation in either domain. (B) An eye disc that contains SerDN flp-out clones. Overexpression of SerDN decreased ventral cell viabilities such that only small ventral clones were observed. By contrast, there were more dorsal clones and they were considerably larger. (C) Overexpression of SerDN by the ey-Gal4 driver abolished eye development. (D) SerDN flp-out clones caused a preferential loss of the ventral domain, similar to L.

View larger version (63K): [in a new window]   Fig. 5. L is a novel protein with preferential expression anterior to the furrow. (A) Overlapping cosmids spanning the 51A2-B1 regions were isolated. Cos D3, but not cos F7, rescued the L phenotype and the lethality of LP17 allele, which has a P-element inserted in the first exon. E: EcoRI site. (B) The putative L protein contains a poly-glutamine rich region (underlined). Arrowhead indicates the P-element insertion site of LP17. (C) Sequences of the C terminus of L and its related proteins over a span of 67 amino acids: bee (BI509118) (43% identical; 63% positive), mouse (AK003638) and human (BC007416) (37% identical; 53% positive). In the last 30 amino acids, 56% are identical. The red letters indicate identical amino acids and green ones indicate similar amino acids. (D) Full-length L cDNA detected a single band on a northern blot of total RNA extracted from larvae of all stages. (E) Embryo of Lrev6-3/Lrev6-3 (arrowhead), a loss-of-function allele, has no detectable level of L protein (red). In Lrev6-3/+ embryo (arrow) the protein was readily detected. The genotype of the embryos was distinguished with green fluorescent protein marker (not shown). (F,G) In third instar eye/antenna discs, L transcripts, detected by RNA in situ hybridization (F), and L protein, detected by antibody (G), are most intense in the outer antenna ring and in the eye disc anterior to the furrow

View larger version (47K): [in a new window]   Fig. 6. Drosophila eye disc is partitioned into three domains. (A) Dorsal cells, ventral midline cells (yellow outline) and ventral cells (green outline) may develop independently. (B) Molecular interactions among N ligands and N in midline cells (yellow cells) result in the non-autonomous activation of L/Ser pathway to promote proliferation of the rest of the ventral domain (green cells).