The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development

Abstract

Inositol 1,4,5-trisphosphate (IP3) regulates a host of biological processes from egg activation to cell death. When IP3-specific receptors (IP3Rs) bind to IP3, they release calcium from the ER into the cytoplasm, triggering a variety of cell type- and developmental stage-specific responses. Alternatively, inositol polyphosphate kinases can phosphorylate IP3; this limits IP3R activation by reducing IP3 levels, and also generates new signaling molecules altogether. These divergent pathways draw from the same IP3 pool yet cause very different cellular responses. Therefore, controlling the relative rates of IP3R activation vs. phosphorylation of IP3 is essential for proper cell functioning. Establishing a model system that sensitively reports the net output of IP3 signaling is crucial for identifying the controlling genes. Here we report that mutant alleles of wavy (wy), a classic locus of the fruit fly Drosophila melanogaster, map to IP3 3-kinase 2 (IP3K2), a member of the inositol polyphosphate kinase gene family. Mutations in wy disrupt wing structure in a highly specific pattern. RNAi experiments using GAL4 and GAL80(ts) indicated that IP3K2 function is required in the wing discs of early pupae for normal wing development. Gradations in the severity of the wy phenotype provide high-resolution readouts of IP3K2 function and of overall IP3 signaling, giving this system strong potential as a model for further study of the IP3 signaling network. In proof of concept, a dominant modifier screen revealed that mutations in IP3R strongly suppress the wy phenotype, suggesting that the wy phenotype results from reduced IP4 levels, and/or excessive IP3R signaling.

Keywords: Cam; GAL80; IP3K1; Ipk2; genetic interaction.

Figure 1

Some key components of IP₃-related signaling in Drosophila. Enzyme names are boxed and an encircled “P” denotes an inorganic phosphate group. IP₃ (inositol 1,4,5-trisphosphate, top center) may undergo the following fates: (1, left) bind to the IP₃-gated calcium channel IP3R (IP3 receptor), causing IP3R to open and release calcium that was sequestered in the ER lumen, (2, top right) be phosphorylated by IP3K2 (IP₃ 3-kinase 2) to form IP₄ (inositol 1,3,4,5-tetrakisphosphate), or (3, bottom right) be more highly phosphorylated by Ipk2 (inositol polyphosphate kinase 2), and subsequently Ipk1 (inositol polyphosphate kinase 1) to yield IP₆. Only forward reactions are shown because previous studies suggested that these reactions predominate in fly cells (Seeds et al. 2004). IP3K1 catalyzes the same reaction as IP3K2 but is not the primary focus of this study, and therefore not shown. Calmodulin (Cam), also not shown, increases IP3K2 activity by binding to the enzyme in a calcium-dependent fashion (Lloyd-Burton et al. 2007). See Introduction for additional molecular details. In this report, we present evidence that IP3K2 is encoded by the wavy (wy) locus, and that a balance between IP3K2 and IP3R functioning is necessary for normal wing morphology.

Figure 2

Mutations in wavy (wy) disrupted wing morphology. (A) Wild type (left) and wy⁷⁴ⁱ flies (right). Note that the wy⁷⁴ⁱ flies obtained from the Bloomington Center (BL#1162) were also mutant for the eye color gene vermillion (v¹). In the most extreme cases, a mutation in wy caused the following three phenotypes: (1) a wave-like buckle (black arrow) at a specific location midway along the costal vein, just distal to the intersection of this vein with the first longitudinal vein; (2) an upturn at the most distal margin of the wing (blue arrow); and (3) a generally shriveled appearance with a pattern. However, wy mutants often displayed only specific subsets of these phenotypes. (B–E) Numerical scoring system reflecting the subsets of wy phenotypes that were observed. (B) Phenotypically wild type wings received scores of “0” (note that none of the individuals from the original mutant strains received a wild type score; see Table 1A). (C) A wing with a costal buckle as the only apparent abnormality (black arrow) received a score of “1”. (D) A wing with a costal buckle and distal upturn was scored as a “2”. (E) A wing with a costal buckle, distal upturn, and overall shriveled appearance received a score of “3”. No other combinations of these three phenotypes were observed throughout our experiments.

Figure 3

IP3K2 is the wavy gene. (A) Image depicts the 11E9-11 region of the X chromosome, after an image generated using the Flybase GBrowse tool (St Pierre et al. 2014). From top, dark blue rectangles represent gene boundaries, with arrowheads indicating directions of transcription. Known transcripts are displayed immediately below gene boundaries, with tan representing coding sequence, gray representing noncoding sequence, lines representing introns, and again arrowheads indicating directions of transcription. Red rectangles indicate sequences deleted in the Df(1)BSC766 and Df(1)Exel6245 stocks. These deletions fail to complement wy¹, wy², and wy⁷⁴ⁱ. On the other hand, the sequences duplicated in the Dp(1;3)DC267 and Dp(1;3)DC268 stocks (light blue rectangles) fully complement all three wy alleles. The unshaded region in the middle of the figure highlights the overlap between all four of these deleted and duplicated sequences; IP3K2 is the only gene that expresses full length transcripts and predicted coding sequences from this shared segment. In further support of IP3K2 being the wavy gene, the wy² allele contains a 5-bp deletion (GenBank accession number KT732029) just downstream of a conserved inositol polyphosphate kinase (Ipk) domain (further magnified image at bottom right, dark brown segment). Rescue and RNAi experiments also indicated that IP3K2 is the wavy gene (see Table 1, C and D). Bottom left, a scale bar for the top, low-magnification portion of the figure panel. (B) Putative amino acid sequence of the four contiguous coding exons that are shared by all known IP3K2 transcript isoforms [i.e., the enlarged, brown exons featured at the bottom right of (A)]. Green box highlights a tryptophan residue that is necessary for calmodulin binding (Lloyd-Burton et al. 2007). Underlined, bold sequence represents a domain that is highly conserved by the Ipk superfamily that includes IP3K enzymes. Orange box highlights a PxxxDxKxG motif, which is a key characteristic of the active site (Lloyd-Burton et al. 2007). Yellow box outlines the location and effect of the wy² mutation: a frameshift that changes the sequence of six amino acids then inserts a premature stop codon.

Figure 4

Identifying the critical stage for IP3K2 function in the developing wing using the GAL4-GAL80^ts system and RNAi. nub-GAL4 Tub-GAL80^ts females were mated to RNAi-IP3K2 males, and their nub-GAL4 Tub-GAL80^ts/+; RNAi-IP3K2/+ progeny were reared at either 29°C to express the RNAi construct at high levels or at 18°C to minimize its expression. Control groups (left) were reared at either 29°C or 18°C for their entire life cycle and experimental groups (right) were initially reared at one temperature or the other, shifted from 18°C to 29°C (solid line) or from 29°C to 18°C (dotted line) during a specific developmental window, then maintained at the second temperature for the remainder of their life cycles. Developmental windows during which temperature shifts were administered (x-axis) were designated based on published descriptions of the fly life cycle (Bainbridge and Bownes 1981; Bate et al. 1993): (1) Emb, embryos; (2) Larv, first through mid-third instar larvae; (3) Wand, late third instar-wandering larvae; (4) Pup, white puparium formation-buoyant (P1–P3); and (5) Meta, metamorphosis from head eversion to meconium stages (P4–P15). y-axis indicates wy phenotype scoring as described in Figure 2, B–E and the Results section. Average wing scores are shown with error bars depicting the standard errors of the means. *, P < 0.05 for the Fisher’s exact tests comparing the marked experimental group to each of the two controls on the far left. (For all unmarked experimental groups, P > 0.05 when tested against one of these two controls, and P < < 0.05 when tested against the other control.) Raw data for this experiment are presented in the Table S2.

Figure 5

Potential models for how IP3K2 affects wing morphology and how mutations in IP3R dominantly suppress the wy phenotype. (A) The strong genetic interaction between wy and IP3R, and what is known about the biochemical functions of their encoded proteins, suggest that wing morphology as assessed in this study may be affected by IP₄ signaling, IP₄-independent IP3R signaling, or an integration of both signals. Question marks indicate uncertainty about the relative importance of these two signals. By extension, the wy phenotype may be caused by (B) reduced IP₄ levels, and/or (C) excessive IP3R activation that triggers IP₄-independent signals (e.g., increased Ca²⁺ release from the ER). (B) If the wy phenotype is caused solely by reduced IP₄ levels, then IP3R would be expected to further inhibit accumulation of IP₄ by inhibiting residual activity of mutant IP3K2 enzyme (“Wavy”), for example, by usurping the IP₃ substrate. A mutant copy of IP3R would be expected to make more IP₃ available to IP3K2, increasing IP₄ formation and suppressing the wy phenotype. Increased levels of IP₃ are shown here due to loss of IP3K2 function. However, this model would hold whether or not IP₃ actually accumulates in the wing discs of wy mutants, because in either case, loss of IP3R function could increase the amount of substrate available to the mutant IP3K2 enzyme. (C) Model if increased IP3R signaling triggers downstream, IP₄-independent events to cause the wy phenotype. Here, IP₃ is assumed to accumulate due to loss of IP3K2 function, as suggested by studies in Drosophila S2 cells (Seeds et al. 2004); this accumulation of IP₃ would be expected to hyperactivate IP3R, increasing calcium release from the ER. A partial loss of IP3R function would reduce this excessive IP3R signaling, suppressing the wy phenotype. (B) and (C) represent extreme models that exclude one factor or the other, but a hybrid model is also possible where both IP₄ signaling and IP₄-independent IP3R signaling play significant roles in wing development.