The ryanodine receptor is essential for larval development in Drosophila melanogaster

Abstract

We have investigated the role of the ryanodine receptor in Drosophila development by using pharmacological and genetic approaches. We identified a P element insertion in the Drosophila ryanodine receptor gene, Ryanodine receptor 44F (Ryr), and used it to generate the hypomorphic allele Ryr(16). An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in Ryr(16) larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of Ryr were also investigated. Ryanodine-sensitive Ca(2+) stores had previously been implicated in phototransduction; to address this, we generated Ryr(16) mutant clones in the adult eye and performed whole-cell, patch-clamp recordings on dissociated ommatidia. Our results do not support a role for Ryr in normal light responses.

Figure 1

Ryr¹⁶ is a deletion in Ryanodine receptor 44F. (A) The first three exons of Ryr (of 27) are represented as rectangles, the filled portions indicating coding and the open portions 5′untranslated region sequences. The inverted triangle marks the position of l(2)k04913, and the region deleted in Ryr¹⁶ is indicated by an open rectangle. (H, HindIII; X, XhoI). (B and C) The muscles of newly hatched Ryr¹⁶ and Ryr¹⁶/CyO larvae are visualized by expressing GFP under control of the Mef2 promoter with the UAS/GAL4 system. GFP fluorescence is shown in pseudocolor. (Scale bars, 10 μm.) (B) Ryr¹⁶ heterozygotes phenotypically resemble wild type (data not shown). (C) The muscles of Ryr¹⁶ larvae appear to have developed normally, but the mutant animals appear smaller and rounder than wild type, and the head is not properly extended.

Figure 2

Ryr mRNA is detected in developing visceral and hypodermal muscles. In situ hybridization was performed on wild-type embryos with DIG-labeled probes made from a partial Ryr cDNA. (A) A stage-11 embryo is shown in a lateral view, with dorsal up and anterior to the left. Expression of Ryr mRNA is strongest in the mesoderm. (B) A stage-14 embryo is viewed from the dorsal perspective, anterior to the left. Ryr expression appears widespread with high levels in hypodermal and visceral muscles.

Figure 3

Muscle function is impaired in ryanodine-treated and Ryr¹⁶ larvae. (A) The average BWC/min was determined for newly hatched larvae of the specified genotype. The wild type, Itp-r¹, Ryr^k04913/CyO-GFP^arm, and Ryr¹⁶/CyO-GFP^arm larvae performed identically in this assay. Ryr^k04913 larvae had a slight, reproducible reduction, Ryr¹⁶ a ≈50% reduction, and Ryr¹⁶/Df(2R)Np3 hemizygotes a 90% decrease in BWC/min. The BWC/min for each genotype was measured at least three times on separate collections of larvae (n = 30), and the error is the SEM. (B) Yeast paste freshly doped with 0.01–100 μM ryanodine was fed to newly hatched Ryr¹⁶/CyO-GFP^arm, and the effect on BWC rates was determined after 30 min. Concentrations of ryanodine 1 μM or less and the ethanol control (not shown) had no significant effect on BWC rates, and concentrations ≥100 μM completely inhibited BWC. For each concentration of ryanodine, the BWC measurement was performed on at least two separate larval samples (n = 30), and the error is the SEM. (C) Larvae scored positive for ingestion if they had a concentrated level of dye in the first section of the midgut. Representative curves are shown for Ryr¹⁶/CyO-GFP^arm (○) and Itp-r¹ (□). Ryr^k04913 larvae (◊) showed only a slight lag in reaching 100% positive compared with controls, but the delay was reproducible in multiple trials. In contrast, the percentage of positive Ryr¹⁶ larvae (▵) typically ranged from 40% to 60% at the end of a 6-h time course. (D) To assay excretion, larvae positive for ingestion after feeding blue yeast for 4 h were transferred to unadulterated yeast paste and scored for the complete loss of dye. Ryr¹⁶/CyO-GFP^arm (○), Itp-r¹ (□), and wild-type larvae (not shown) had nearly identical excretion time courses. The rate of excretion was significantly decreased in Ryr¹⁶ larvae (▵). For C and D, each larval genotype was assayed in at least three independent trials (n ≥ 30).

Figure 4

Dorsal vessel contraction is reduced in Ryr¹⁶ and ryanodine-treated larvae. (A) GFP is expressed in all muscles of a wild type, second instar larvae (see Fig. 2). (B) The cardial cells of the heart are shown at higher magnification with the dorsal vessel in the closed position. (Scale bars in A and B, 10 μm.) (C) Heart rate was measured by counting the number of beats per minute for a population of each larval genotype. There was no significant difference between the heart rate of wild-type (second instar) and Ryr¹⁶/CyO (first instar) larvae. Both Ryr¹⁶ larvae and second instar larvae treated with 100 μM ryanodine, however, showed a marked reduction in the rate of contraction. Heart rates were measured for each genotype in at least three independent experiments (n ≥ 15), and the error is the SEM.

Figure 5

Phototransduction is normal in Ryr¹⁶ eye clones. (A) Representative whole-cell voltage-clamp recordings of light-induced currents from a wild type (wt) and a Ryr¹⁶ photoreceptor. (Left) Cells were stimulated with 10-ms flashes of increasing light intensity given at the arrow. Numbers refer to the log order of light intensity associated with that light response (e.g., −4 is 10 times less light than −3). (Right) Cells were stimulated with a 400-ms pulse of light (log [I] = −1). In each case, Ryr¹⁶ responses are indistinguishable from wild type. (B) Quantitative analysis of responses to 10-ms flashes of light. The plot shows peak response amplitude vs. light intensity for wild type and Ryr¹⁶, and the bar graphs compare rise time (measured as the time between 10–90% rise of the peak current) and decay time (measured as the time between 90–10% decline of the current) vs. light intensity. Black bars correspond to wild type, white to Ryr¹⁶. Each data point represents the mean response ± SE for seven wt cells and 15 Ryr¹⁶ mutant cells sampled from 12 Ryr¹⁶ mutant patches.