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First published online 16 October 2008
doi: 10.1242/dev.026773

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The development of motor coordination in Drosophila embryos

¹ Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.
² Lehrstuhl für Genetik und Neurobiologie, Theodor-Boveri-Institut Biozentrum, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Recording and analysing muscle contractions in freely moving Drosophila larvae. (A) Four images from a movie of a first-instar larva during forward crawling (a wave of muscle contractions that propagate from posterior to anterior, propelling the larva forward). Images are enlarged and selected to show onset of contraction (i,ii) and beginning of relaxation (iii,iv) in muscles of segment A5, during the sequence. (B) Entire sequence (two waves of forward peristalsis showing all abdominal segments) analysed frame-by-frame, with onsets and offsets of ventral longitudinal muscle contractions on either side of the animal in segments A1-7 documented.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Gradual development of coordinated sequences. Example data from continuous recording of movements in a single embryo 16-19 hours AEL, using muscle imaging. Before 17 hours AEL, muscle contractions occur as isolated twitches and unilateral rapidly propagated sequences across segments (arrowheads), which in intact embryos cause rolling within the vitelline membrane. At 17 hours AEL, there is a burst of activity (brief repeated muscle contractions occurring asynchronously in all abdominal segments, with little side-to-side coordination). Later bursts of activity contain motifs resembling elements of forward or backward crawling, in that muscles on left and right sides contract together, and, in comparison with earlier stages, contractions are relatively prolonged (arrowhead). At 18.25 hours AEL, embryos begin to perform sequences resembling complete waves of larval crawling, together with partial waves typically seen slightly earlier in development (arrowheads).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Locomotor development in wild-type Drosophila embryos. Movements recorded from three embryos imaged from 16 hours AEL until hatching (around 21 hours AEL) using muscle imaging. From 17 hours AEL, muscle activity is episodic: sustained (30 seconds-2 minutes) bursts of muscle contraction are separated by longer periods of relative quiescence. The first complete wave of forward peristalsis occurs at 18.25 hours AEL and the traces are aligned at this point. Black bars indicate hatching.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Acquisition of larval reflexes in prematurely hatched Drosophila embryos. (A) Touch response. Average touch response scores are shown (and s.e.m.). Each embryo was tested 10 times (giving a score out of 40) and 10 embryos were tested at each developmental age. Embryos were selected at tracheal filling (18.5 hours AEL) and aged on agar plates at 25°C, then prematurely hatched just before testing). (B) Acquisition of self-righting. Embryos were rolled upside down onto their dorsal surface, and time to self-right recorded. Embryos were tested three times and 10 embryos were tested at each developmental age (embryos at different developmental stages were selected as in A).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Timeline of behavioural and morphological development. Timeline showing major changes in contraction patterns identified by muscle imaging, and onset of larval-like reflexes. Morphological development 16-18.5 hours AEL [roughly corresponding to stages 17b-d of Pereanu et al. (Pereanu et al., 2007)]. At 16.0 hours AEL, spiracles are detected as dorsal papillae on the terminal segment. At 16.5 hours, uric acid is detected in the Malpighian tubules, becoming strong by 17.0 hours. At 17.5 hours the median tooth tip becomes faintly visible anteriorly. By 18.0 hours the tooth is clear but tracheae have not filled. At 18.5 hours, tracheae fill.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Patterns of muscle contraction in embryos with and without synaptic transmission. (Left) Muscle contractions recorded at 15-minute intervals from an embryo that lacks all evoked synaptic transmission through pan-neuronal expression of active tetanus toxin (elav-GAL4;UAS-TNT-G). (Right) Similar recording made from a control embryo expressing inactive tetanus toxin (elav-GAL4;UAS-TNT-VIF). For each genotype, four embryos were recorded and analysed. Similar data were obtained in each case, but for clarity, results from single embryos are shown. Arrow indicates the first complete wave of forward peristalsis.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Disrupting glutamate-mediated transmission at the NMJ reduces myogenic movements in the embryo. (A) Representative data for an embryo expressing grim panneuronally (elav-GAL4;UAS-grim) and an embryo homozygous for a mutation in an essential glutamate receptor subunit GluRIII. Unilateral waves of contraction occur in both, persisting for several hours. (B) Mean proportion of time each VL muscle spent in contraction 17-18 hours AEL is calculated for each genotype (n=4 for each genotype, with four or five 2-minute traces analysed for each embryo, error bars indicate s.e.m.). Embryos mutant for GluRIII show a lower frequency of contractions than embryos in which transmission at the NMJ is blocked presynaptically (through expression of TNT-G), and embryos lacking presynaptic terminals (through expression of grim).

View larger version (39K): [in this window] [in a new window]   Fig. 8. Patterns of muscle contraction with and without sensory input. (Left) Muscle contractions recorded at 15-minute intervals from an embryo without sensory input (PO163-GAL4;UAS-TNT-G). (Right) A similar recording made from control embryo with normal synaptic transmission from sensory neurons (PO163-GAL4;UAS-TNT-VIF). For each genotype, five embryos were recorded and analysed, but results from single representative embryos are shown. In embryos with no sensory input, a burst of muscle contractions occurred 17 hours AEL, as in controls. However, the first properly coordinated sequences are delayed in embryos that lack sensory input (arrows).

View larger version (28K): [in this window] [in a new window]   Fig. 9. Activity-dependent depression during bursting motor output. (A) (i) Mean level of activity before and after bursts in wild-type embryos (n=3, 15 pre-burst and 15 post-burst sequences) (error bars=s.e.m.). (ii) Number of muscle contractions (in 1-minute time bins) from start of second burst until the end of third burst in five embryos. Data are normalised so that each cycle duration=1. (iii) Regression lines for number of contractions per minute during inter-burst interval. (B) Activity-dependent depression in embryos expressing channel rhodopsin 2 in all neurons (elav-GAL4;UAS-ChR2). Average response scores are shown for different intervals from the end of a naturally occurring burst of activity (n=5 at each time interval and error bars indicate s.e.m.). Response scores correspond to shortest stimulus pulse producing a long-lasting (over 15 seconds) vigorous contractile response (response to 25 mseconds scores 5, 50 mseconds 4, 100 mseconds 3, 200 mseconds 2, 400 mseconds 1, no response to any pulse 0). Stimuli: 488 nm light and control wavelengths that do not activate ChR2 (568 and 638 nm).

View larger version (11K): [in this window] [in a new window]   Fig. 10. Activity-dependent depression during bursting motor output. (A) (i) Mean level of activity before and after bursts in wild-type embryos (n=3, 15 pre-burst and 15 post-burst sequences) (error bars=s.e.m.). (ii) Number of muscle contractions (in 1-minute time bins) from start of second burst until the end of third burst in five embryos. Data are normalised so that each cycle duration=1. (iii) Regression lines for number of contractions per minute during inter-burst interval. (B) Activity-dependent depression in embryos expressing channel rhodopsin 2 in all neurons (elav-GAL4;UAS-ChR2). Average response scores are shown for different intervals from the end of a naturally occurring burst of activity (n=5 at each time interval and error bars indicate s.e.m.). Response scores correspond to shortest stimulus pulse producing a long-lasting (over 15 seconds) vigorous contractile response (response to 25 mseconds scores 5, 50 mseconds 4, 100 mseconds 3, 200 mseconds 2, 400 mseconds 1, no response to any pulse 0). Stimuli: 488 nm light and control wavelengths that do not activate ChR2 (568 and 638 nm).

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