(A) Third instar larvae at 120 hrs After Egg Laying (AEL). UASitpr⁺/+;itpr^sv35/ug3 are significantly reduced in size and fat body content. Both Dilp2GAL4 and DdcGAL4 rescued larvae start wandering at this stage and appear similar to wild-type (Canton-S) controls. (B) Wild-type (Canton-S) and animals of all rescue conditions grown at 25°C survive better as compared to itpr mutants (UASitpr⁺/+;itpr^sv35/ug3) grown under the same conditions at later times after egg laying (*p<0.05; Student's t-test). For each time interval and genotype, 75 animals were screened in 3 batches of 25. Each bar represents the total viability of the indicated genotype. The colored subdivisions in each bar represent the number of larvae developing to later larval, pupal (P) or adult (A) stages. The survival profile of DdcGAL4 rescued animals (UASitpr⁺/+; DdcGAL4/+; itpr^sv35/ug3) is better than that of Dilp2GAL4 rescued condition (UASitpr⁺/+; Dilp2GAL4/+; itpr^sv35/ug3). Expression of UASitpr⁺ simultaneously with Dilp2GAL4 and DdcGAL4 (UASitpr⁺/+; Dilp2GAL4/DdcGAL4; itpr^sv35/ug3) does not improve survival beyond that observed with only DdcGAL4. Results are expressed as mean±SEM.

(A, C) At 60 hrs and 108 hrs AEL, itpr^sv35/ug3 have much less red food in their guts in comparison to UASitpr⁺/+;DdcGAL4/+;itpr^sv35/ug3, UASitpr⁺/+;Dilp2GAL4/+; itpr^sv35/ug3 and wild-type larvae. (B, D) Spectrophotometric quantification of homogenates from larvae fed yeast paste containing a red dye. Control, Dilp2GAL4 or DdcGAL4 rescued larvae ingest significantly more dye than itpr^sv35/ug3 (itpr mutant) larvae at 60 hrs AEL (*p<0.05; Student's t-test) and at 108 hrs AEL (*p<0.005; Student's t-test). The following number of larvae (n) in batches (N) were assayed for each genotype: At 60 hrs AEL: n = 95 or more, N = 4 for all genotypes; at 108 hrs AEL: for UASitpr⁺/+;itpr^sv35/ug3 L3 n = 46, N = 3; L2 n = 87, N = 3; for all other genotypes n = 100, N = 4 or more. Quantification of cell number in salivary glands from larvae at 60 hrs AEL stained with DAPI to visualize nuclei. itpr^sv35/ug3 (itpr mutant) in (E) and wild-type are shown in (F). No significant difference (G) was observed in the number of nuclei in itpr mutant and wild-type salivary glands. n = 10 salivary glands for each genotype. RT-PCR analysis (H) and quantitative real-time PCR analysis (I, J) revealed significant up-regulation of transcript levels of d4E-BP and dLipase-3 in itpr^sv35/ug3 at 60 hrs AEL that can be significantly rescued by Dilp2GAL4 or DdcGAL4 driven expression of UASitpr⁺ (*p<0.005; Student's t-test). Real-time PCR analysis was repeated three times with independently isolated RNA samples for each genotype. Results are expressed as mean±SEM.

(A) From left to right: UASitpr⁺/+;Dilp2GAL4/+; P0163GAL4,itpr^sv35/ug3 (Dilp2GAL4 P0163GAL4 rescue), wild-type (Canton-S) and UASitpr⁺/+;Dilp2GAL4/+;itpr^sv35/ug3 (Dilp2GAL4 rescue) pupae. (E) Only UASitpr⁺ expression with Dilp2GAL4 in itpr^sv35/ug3 background causes a significant increase (*p<0.005; Student's t-test) in pupal length as compared to controls of all the indicated genotypes. Pupal length is restored close to wild-type in Dilp2GAL4::P0163GAL4 rescue condition. (B) From left to right: wild-type and Dilp2GAL4 rescued flies. (F) Body length of Dilp2GAL4 rescued flies is significantly more than wild-type flies (*p<0.05; Student's t-test). (C, D) From left to right: UASitpr⁺/+;DdcGAL4/+;P0163GAL4,itpr^sv35/ug3 (DdcGAL4 P0163GAL4 rescue), wild-type (Canton-S) and UASitpr⁺/+;DdcGAL4/+;itpr^sv35/ug3 (DdcGAL4 rescue) pupae (C) and flies (D). (G) Only UASitpr⁺ expression with DdcGAL4 in itpr^sv35/ug3 background causes a significant increase (*p<0.005; Student's t-test) in pupal length as compared to controls of all the indicated genotypes. Pupal length (G) and adult weight per fly (H) is increased in the DdcGAL4 rescued condition (*p<0.005; Student's t-test) but is restored close to wild-type in DdcGAL4::P0163GAL4 rescue condition. n = 10 or more for each individual genotype for (A–G). For (H), the following numbers of male flies (n) in batches of around 3 flies each were weighed for each genotype: DdcGAL4 P0163GAL4 rescue n = 26, wild-type n = 30 and DdcGAL4 rescue n = 54. (I) Total number of larvae that undergo pupation is significantly increased on introducing a prothoracic gland GAL4 (P0163GAL4) in DdcGAL4 and Dilp2GAL4 rescued conditions (*p<0.05, **p<0.005; Student's t-test). (J) Time AEL for 50% pupal formation is significantly reduced with P0163GAL4 in DdcGAL4 and Dilp2GAL4 rescued conditions (*p<0.005; Student's t-test). However the 50% pupation time in all single and double GAL4 conditions remained longer than the 50% pupation time of wild-type. For (I) and (J) 25 larvae in the following number of batches (N) were assayed for pupation for each genotype: wild-type N = 11, Dilp2GAL4 rescue N = 10, Dilp2GAL4 P0163GAL4 rescue N = 5, DdcGAL4 rescue N = 13 and DdcGAL4 P0163GAL4 rescue N = 9. (K) DdcGAL4 rescued itpr^sv35/ug3 larvae pupated earlier on being fed 20-hydroxyecdysone (∼150 hrs AEL) than larvae without 20-hydroxyecdysone (∼200 hrs AEL) (*p<0.05; Student's t-test). A minimum of 75 animals were screened in batches of 25 each. Differences in pupation rate are not apparent upon 20-hydroxyecdysone feeding in Dilp2GAL4 rescue animals due to increased lethality in this condition in late third instar larvae. The DdcGAL4 rescued condition which were not fed ecdysone, pupated at a slower rate than those observed in (J). This is very likely due to differences in culture conditions in the two cases. Results are expressed as mean±SEM.

(A) A schematic drawing depicting a third instar larval brain with the relative positions of the IPCs and their processes (in green) and the Ddc labeled cells and their processes (in red). The cellular processes from the two domains seem to intermingle in the sub-esophageal ganglia region. (B–H) Three-dimensional projections of confocal Z-stacks of a wild-type Drosophila larval brain from a wandering third instar larva expressing mCD8GFP with Dilp2GAL4 and immunostained with anti-serotonin antibody (E), anti-Ddc antibody (B, F) and anti-GFP antibody, (C, G). (D) is a merge of (B) and (C) while (H) is a merge of (E),(F) and (G). In (D) and (H), anti-Ddc staining is in red and anti-GFP in green while anti-serotonin is blue in (H). Red arrowheads in (B, F) indicate Ddc stained cells in the sub-esophageal ganglia that lie in close proximity to IPC projections (bottom green arrows in C, G). Smaller red arrowheads indicate cells which send out processes (marked with red arrows) that seem to intermingle with these IPC projections. Green arrowheads in (C, G) mark the IPCs in the two brain lobes. Green arrows indicate the projections of the IPCs towards the lateral protocerebrum (top green arrows) and sub-esophageal ganglion (bottom green arrows). Ddc marked cells (indicated by big red arrowheads in B, F) stain with the anti-serotonin antibody (E, marked by blue arrowheads), but have lesser serotonin staining than some neighboring cells (for example, cells in the lateral protocerebrum indicated by blue asterisk in E). Scale bars B–H 20 µm.

Three-dimensional projections of confocal Z-stacks of a wild-type Drosophila 3^rd instar larval brain (A–C) expressing mCD8GFP with DdcGAL4 and immunostained with an anti-Ddc antibody (A) and anti-GFP antibody (B). A majority of DdcGAL4::UASmCD8GFP labeled cells overlap with those stained with the Ddc antibody, though there are some cells in both cases that do not overlap (green arrows in B). These do not appear in the region of the IPCs. Green arrowhead in (B) indicates DdcGAL4::UASmCD8GFP expression in Ddc stained cells in the sub-esophageal ganglia that lie in close proximity to IPC projections. Scale bars A–C, 50 µm.

(A) Act5c dsitpr larvae (Act5cGAL4/UASdsitpr¹⁰⁶³) did not undergo pupation and died as 3^rd instars. The number of pupae formed in Dilp2 dsitpr (Dilp2GAL4/UASdsitpr¹⁰⁶³; UASdicer/+), Ddc dsitpr (DdcGAL4/UASdsitpr¹⁰⁶³; UASdicer/+) and C155 dsitpr (Elav^C155GAL4; UASdsitpr¹⁰⁶³/+;UASdicer/+) was similar to controls (not significant; p>0.05; Student's t-test). GFP positive larvae were used as controls in each RNAi experiment. Three batches of 25 2^nd instar larvae were screened for each of the indicated genotypes. Results are expressed as mean±SEM. (B) Significant reduction in larval size was observed in Actin5cGAL4/UASdsitpr¹⁰⁶³ 3^rd instars (∼120 hrs AEL) compared to controls (Actin5cGAL4 or UASdsitpr¹⁰⁶³/CyoG). (C) RT-PCR gel with up-regulation of dLipase-3 transcript levels in RNA isolated from Actin5cGAL4/UASdsitpr¹⁰⁶³ larvae (∼ 85 hrs AEL) compared to RNA from controls. (D) A western blot with reduced InsP₃R (280Kda) protein levels in lysates from Actin5cGAL4/UASdsitpr¹⁰⁶³ 3^rd instar (∼120 hrs AEL) larvae as compared to lysates from controls. Equal levels of the loading control α-spectrin (278Kda) confirm that similar quantities of protein lysates were loaded in each lane.