Distinct Effects of Abelson Kinase Mutations on Myocytes and Neurons in Dissociated <i>Drosophila</i> Embryonic Cultures: Mimicking of High Temperature

<div><p>Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction <i>in vivo.</i> The <i>Drosophila</i> primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in <i>Abl</i> mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, <i>Abl</i> myocytes frequently displayed well-expanded lamellipodia. Conversely, <i>Abl</i> neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (<i>Abl/Abl</i> vs. <i>Abl</i>/+) of different alleles (<i>Abl¹ and Abl⁴</i>) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of <i>Abl</i> cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic <i>Abl</i> neuronal morphology. Intriguingly, HT incubation also promoted <i>Abl</i> lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development typical of <i>Abl</i> cultures. Despite the extensive alterations by <i>Abl</i> mutations, we observed myocyte fusion events and nerve-muscle contact formation between WT and <i>Abl</i> cells in mixed WT and <i>Abl</i> cultures derived from labeled embryos.</p></div

Comparison of the Abelson Kinase mutation and high-temperature effects on neuronal growth.

<p>Phase contrast images (100X) of stage 10 WT and <i>Abl</i> cultures grown at room temperature (RT) or high temperature (HT, 30°C). A1 vs. B1) In RT cultures, <i>Abl</i> neurons show enhanced growth of filopodia compare to WT. There is also an increase in dark nodules along the neurite and at the terminal (B1, arrowheads). However, while phase-light growth cones with expanded lamellipodia are rare in <i>Abl</i>, they can readily be seen in WT (A1, arrow). A1 vs. A2) HT incubation of WT neurons produces extremely large growth cones (A2, arrows). B1 vs. B2) HT incubation of <i>Abl</i> neurons enhances development of phase-light lamellipodia at growth cones and along neurites (B2, arrows). All cultures were incubated 3–6 days. Scale bars, 10 µm.</p

Analysis of WT and <i>Abl</i> muscle growth at RT and HT.

<p>A) Muscle cell numbers per single-embryo culture from stage 10 WT and <i>Abl</i> embryos incubated at RT and HT. At RT, abundant muscle cells were present in <i>Abl</i> cultures but nearly absent in WT cultures (***p<0.001, rank test). HT-incubation greatly increased the number of muscle cells in WT cultures (***p<0.0001, rank test) while exerted little effect on <i>Abl</i> cultures. Similar proportions between muscle cells isolated from cell clusters and those still associated with clusters were observed among HT WT cultures, RT <i>Abl</i> cultures, and HT <i>Abl</i> cultures (57%, 41%, and 57% isolated muscle cells, respectively, for the 3 cultures). Error bars, SEM. Sample sizes for A (number of coverslips (C) and number of muscle cells (N) for each culture): WT RT, C = 17, N = 20; WT HT, C = 6, N = 633; <i>Abl</i> RT, C = 6, N = 238; <i>Abl</i> HT, C = 7, N = 371. Note that muscles cells in WT RT cultures were nearly absent (arrows). B) Average number of lamellipodia per muscle cell. The number for <i>Abl</i> RT was greater than both WT RT and WT HT. C) Increased degree of muscle fusion by HT incubation and <i>Abl</i> mutations. A majority of muscle cells in each culture type were multinucleated, except for WT RT cultures, in which very few muscle cells were encountered (total 20 in 17 cultures) and they were exclusively mononucleated (arrows). The fusion rate is significantly increased after HT incubation (WT, p<0.0001; <i>Abl,</i> p<0.03, χ² test). The identical cell samples from the same cultures were analyzed for B & C (number of muscle cells (N) and number of lamellipodia (n): WT RT, N = 20, n = 0; WT HT, N = 296, n = 39; <i>Abl</i> RT, N = 228, n = 60; <i>Abl</i> HT, N = 92, n = 44). Same cultures shown in A were analyzed. All cultures were derived from stage 10 embryos and incubated 2–5 days.</p

Nerve-muscle interactions between WT and <i>Abl</i> cells.

<p>A1 & B1) Merged images of phase contrast and fluorescent optics. The cells labeled red are from Da-Gal4>UAS-RFP embryos (<i>Abl</i>^+/+) and the unlabeled cells are from <i>Abl</i> embryos. A1) Neurites from an <i>Abl</i> neuronal cluster interact with a group of <i>Abl</i> and <i>Abl</i>^+/+ muscle cells, which are potentially in the process of fusion. A2) Enlargement of the boxed area in A1 showing nerve-muscle contact. B1) <i>Abl</i> neurites contact a multinucleated muscle cell, which is labeled by RFP. B2) Enlargement of boxed area in B1. This suggests that <i>Abl</i> neurons can make contact with WT muscle cells (and vice versa, data not shown). Age of culture, 3 days. Cultures were derived from stage 10 <i>Abl</i> mutants and stage 12 WT embryos. Scale bar, 10 µm.</p

Promotion of muscle lamellipodia development and interaction with neurons in <i>Abl</i> cultures.

<p>A–D) Samples of phase contrast images (100X) displaying lamellipodia from WT and <i>Abl</i> muscle cells in culture. (A) is from cultures incubated 7D, (B–D) are from cultures incubated 24 h–48 h. WT cultures from advanced embryos (stage 12) produce expanded lamellipodia when incubated at RT (A), whereas cultures derived from earlier embryos (stage 10) show muscle cells only after HT incubation, which display microspikes but rarely well extended lamellipodia (B). <i>Abl</i> cultures exhibit more abundant lamellipodia at stage 10 following incubation at RT (C) or HT (D). E) Example of neuron-muscle interactions in HT-incubated <i>Abl</i> cultures. Time lapse images (phase contrast, 40X) taken 24 hours (E1) and 39 hours (E2) after plating. Neuronal filopodia and muscle lamellipodia first approached each other (E1) and subsequently formed morphological connection (E2). Arrows indicate the interaction site. Scale bars, 10 µm.</p

High-temperature incubation promotes muscle development in cultures from stage 10 WT embryos.

<p>A) WT cultures from stage 10 embryos rarely presented any muscle cells when incubated at RT (40X). B1) HT incubation of stage 10 WT cultures produced abundant muscle cells both in isolation and in association with cell clusters. Sample from culture after 39 h incubation at HT (40X), showing a muscle cell isolated from cell aggregations (arrow) and still others associated with cell clusters (arrowheads). B2–B3) WT cultures after 9 days incubation at HT (100X). B2) An unusually large WT multinucleated muscle cell (arrows pointing to nuclei) interacting with neurites from a neuron (soma not shown). B3) Enlarged image of boxed area in B2 displaying a neuromuscular contact. B2, phase contrast; A, B1, and B3, DIC images. Scale bars, 20 µm.</p

Mitochondria are highly enriched in the dark nodules along neurites of <i>Abl</i> neurons.

<p>A1–B1) Phase contrast images of phase dark nodules and phase light lamellipodia from WT (RT) and <i>Abl</i> (HT) neutires (100X). A2–B2) Merged fluorescent and phase contrast images showing locations of Rh123 staining. Arrowheads, phase light growth cones. Arrows, phase dark structures along the neurite that accumulate Rh123 staining. The staining indicates that dark nodules in <i>Abl</i> are enriched with mitochondria and possibly other organelles. Age of cultures, 2–4 days. All cultures were derived from stage 10 embryos. Scale bar, 10 µm.</p

Sample of muscle cell elongation and fusion in HT-incubated WT culture.

<p>Time lapse phase contrast images (100X) taken 39 hours (A) and 46 hours (B1) after plating. Arrows denote an example of muscle elongation. B2) Enlarged DIC image of the boxed area in B1. Arrowhead indicates a potential fusion site. All cultures were derived from stage 10 embryos. Scale bars, 10 µm.</p

A Novel Role for Ecdysone in <i>Drosophila</i> Conditioned Behavior: Linking GPCR-Mediated Non-canonical Steroid Action to cAMP Signaling in the Adult Brain

<div><p>The biological actions of steroid hormones are mediated primarily by their cognate nuclear receptors, which serve as steroid-dependent transcription factors. However, steroids can also execute their functions by modulating intracellular signaling cascades rapidly and independently of transcriptional regulation. Despite the potential significance of such “non-genomic” steroid actions, their biological roles and the underlying molecular mechanisms are not well understood, particularly with regard to their effects on behavioral regulation. The major steroid hormone in the fruit fly <i>Drosophila</i> is 20-hydroxy-ecdysone (20E), which plays a variety of pivotal roles during development via the nuclear ecdysone receptors. Here we report that DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in <i>rutabaga</i> (Ca²⁺/calmodulin-responsive adenylate cyclase) mutants was rescued by <i>DopEcR</i> overexpression or acute 20E feeding, whereas a memory defect in <i>dunce</i> (cAMP-specific phosphodiestrase) mutants was counteracted when a loss-of-function <i>DopEcR</i> mutation was introduced. A memory defect caused by suppressing dopamine synthesis was also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB)—a brain region central to learning and memory in <i>Drosophila</i>—is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. Our multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids.</p></div

DopEcR is required for the 30-minute courtship memory induced by 1-hour courtship conditioning.

<p>(A) Thirty-minute courtship memory in wild-type flies (control) and flies heterozygous (<i>DopEcR^PB1</i>/+), homozygous (<i>DopEcR^PB1</i>/<i>DopEcR^PB1</i>), and hemizygous (<i>DopEcR^PB1</i>/Df(3L)ED4341) for <i>DopEcR</i>. <i>DopEcR^PB1</i> homozygotes and hemizygotes were defective for 30-minute courtship memory. (B) Time course of courtship memory in <i>DopEcR^PB1</i> homozygotes. Significant memory was observed immediately after conditioning, but not 15 or 30 minutes after conditioning. (C) A defect in 30-minute courtship memory in flies that ubiquitously express the <i>DopEcR</i> RNAi after eclosion, in response to RU486 stimulation of the <i>tub</i>-GS-Gal4 driver. The presence or absence of courtship memory was evaluated by applying the Mann–Whitney U-test to naïve and conditioned males. Statistical significance is shown above each bar as NS, no significant difference, **, <i>P</i><0.01 or ***, <i>P</i><0.001. Sample numbers for naïve and conditioned flies are shown under each graph. PIs were analyzed using Krustal-Wallis One-Way ANOVA, followed by Dunn's pairwise test for multiple comparisons. #, <i>P</i><0.05; ##, <i>P</i><0.01. Error bars (s.e.m.).</p