Ca2+-imaging and photo-manipulation of the simple gut of zebrafish larvae in vivo

Zebrafish larval gut could be considered as an excellent model to study functions of vertebrate digestive organs, by virtue of its simplicity and transparency as well as the availability of mutants. However, there has been scant investigation of the detailed behavior of muscular and enteric nervous systems to convey bolus, an aggregate of digested food. Here we visualized peristalsis using transgenic lines expressing a genetically encoded Ca2+ sensor in the circular smooth muscles. An intermittent Ca2+ signal cycle was observed at the oral side of the bolus, with Ca2+ waves descending and ascending from there. We also identified a regular cycle of weaker movement that occurs regardless of the presence or absence of bolus, corresponding likely to slow waves. Direct photo-stimulation of circular smooth muscles expressing ChR2 could cause local constriction of the gut, while the stimulation of a single or a few neurons could cause the local induction or arrest of gut movements. These results indicate that the larval gut of zebrafish has basic features found in adult mammals despite the small number of enteric neurons, providing a foundation for the study, at the single-cell level in vivo, in controlling the gut behaviors in vertebrates.


Results and discussions
In double transgenic fish, SAGFF(LF)134A; Tg(UAS: GFP), GFP was expressed in the gut at the distal intestine and the posterior end of the middle intestine after 2 dpf 16 (Fig. 1a-c). We found that GFP-positive cells were immunolabeled with anti-Desmin antibody, a marker for smooth muscles 9 (Fig. 1e). We discerned a single cell by using a photoconversion technique 17 with SAGFF(LF)134A; Tg(UAS: Kaede) in 5 dpf larvae. When we irradiated a single cell with a 405 nm laser, it was successfully labeled with red fluorescence in the local area (Fig. 1d). The photoconverted cell was ribbon-shaped, i. e., elongated and flat; it wrapped around the gut in perpendicular to the axis of the gut and had tapered ends (Fig. 1d). These data indicate that expression was mainly in the circular smooth muscles, while expression was also observed in some larvae in a small number of longitudinal muscles (Fig. 1e, lower panel).
We first visualized circular smooth muscle activity using the genetically encoded Ca 2+ sensor GCaMP3 18,19 . To monitor changes in the intracellular Ca 2+ concentration in circular smooth muscles of the gut during peristaltic  10 . The PI is also referred to as the intestinal bulb and is thought to have similar functions as the stomach, which zebrafish do not have. The MI is thought to absorb nutrients and plays a role in mucosal immunity. The DI is analogous to the colon. M mouth, OC oral cavity, Ph pharynx, E esophagus, PI proximal intestine, MI middle intestine, DI distal intestine, A anus. (b) A higher-magnification image of the area indicated by the square in (a). Scale bar, 50 μm. (c) Lateral view of SAGFF(LF)134A; Tg(UAS: GFP) at 8 dpf. Scale bar, 200 μm. (d) Live confocal image of individual circular smooth muscles visualized by green to red photoconversion of Kaede fluorescence in SAGFF(LF)134A; Tg(UAS: Kaede) at 5 dpf. Lateral projection view shows two muscles photoconverted. Left side view. Cross-slice view of the dotted area shows a single red muscle. Scale bar, 10 μm. (e) Saggital sections of a confocal image of SAGFF(LF)134A; Tg(UAS: GFP) at 8 dpf stained with anti-Desmin antibody. Upper panels demonstrate Desmin filaments (in magenta, shown by triangles) located inside of each circular smooth muscles expressing GFP. Lower panels show an example of occasional appearance of longitudinal muscle cell expressing GFP, containing Desmin filaments (arrows). Scale bar, 10 μm. (f) Saggital section and cross-slice of the gut, indicating a variety of cell types visualized at 5 dpf in Tg(hsp70: Gal4); Tg(UAS: GFP) by heat shock at 3 dpf. Double immunostaining for anti-GFP (green) and anti-5-HT (magenta). EC serotonergic endochromaffin cells, MC mucosal cells, L lumen, CM circular muscle, SN neurites of serotonergic neurons. Scale bar, 50 μm. (g) Schematic diagram showing the hypothetical circuitry for the peristaltic reflex in the zebrafish gut. 1, endochromaffin cell; 2, intrinsic primary afferent cell; 3, ascending interneuron; 4, excitatory motoneuron; 5, 9, interstitial cells of Cajal; 6, 10, circular smooth muscles; 7, descending interneuron; 8, inhibitory motoneuron. www.nature.com/scientificreports/ www.nature.com/scientificreports/ movement, we fed larvae with paramecium from 5 dpf and performed Ca 2+ imaging of the gut at 8 dpf of SAGFF(LF)134A; Tg(UAS: GCaMP3) from the lateral side. We found strong Ca 2+ events in the circular smooth muscles during peristaltic movement in 7 out of 10 larvae. Figure Figure 2d shows kymographs of the GCaMP3 fluorescence and a bright field image, as well as the time course of intensity at each site, denoted by the five squares in Fig. 2b. The fluorescence intensity was stronger at the oral side of the bolus and weaker at the anal side. There was often a sharp boundary between the two sides (arrow in Fig. 2c). Strong Ca 2+ events occurred repeatedly, typically starting at the oral side of the bolus. These Ca 2+ events moved to the anal side (a movement tentatively called a descending wave: DW) and spread to the oral side (ascending wave: AW). In some cases AW was not obviously observed, especially when two or more boluses were in close proximity to each other. DW occurred along with propulsion and expulsion of the bolus. On the other hand, AW may reinforce the power that propels the bolus by adding more area of contraction on the oral side. Most samples showed DW (14/22 events, 7 larvae) and AW (14/22 events, 7 larvae) contractions. When the passage of the bolus was blocked at the anus by agarose gel, in an artificial condition of constipation, we found repeated strong Ca 2+ events (Fig. 3, Supplementary video 2). We calculated the duration and interval of each Ca 2+ event (Fig. 2e,f). The duration (from 50% rise to 50% decay) of the Ca 2+ events ranged from 9.6 to 106.2 s with a mean ± standard deviation (s.d.) of 40 ± 22 s (n = 87, 7 larvae). The mean interval between Ca 2+ events at the oral side of the bolus was 175 ± 105 s (n = 15, 4 larvae).
The activity of other cell types associated with gut movement was also investigated by using Tg(hsp70: Gal4) 20 ; Tg(UAS: GCaMP3) (Fig. 3, Supplementary video 2). Heat shock at 6 dpf induced the mosaic expression of GCaMP3 at 8 dpf in a variety of cell types. The cells that have a round soma and long processes are putative enteric neurons, the cells elongated perpendicularly with gut axis are putative circular smooth muscles. Timelapse observation was also performed on the larva whose bolus was blocked by agarose. We identified two phases of activity in which a population of neurons was synchronously activated or inactivated with muscles involved in peristaltic movement (Fig. 3). Further investigation is needed to identify the neuronal types or other cell types and their connections in the ENS.   . Regular movements observed in the distal intestine at 4 and 7 dpf and peristaltic reflex-like activity observed at 7 dpf without feeding. Kymgraphs of the bright field and GCaMP3 fluorescence movies, at 4 dpf (a) and 7 dpf (b). Regular movements (white triangles) were not always associated with Ca 2+ events in the circular smooth muscles, although weak Ca 2+ events could be sometimes associated with the regular movements, as indicated by asterisks in (a). On the other hand, peristaltic reflex-like movement (magenta triangles) which was sometimes observed without feeding was associated with strong Ca 2+ events in the circular smooth muscles. www.nature.com/scientificreports/ The kymograph analysis of the gut movement, in the bright field in both fed and unfed larvae, demonstrated weaker movement with regular and high frequency, whether with bolus ( Fig. 4) or without (Fig. 5). Ca 2+ signals in the circular muscles associated with this movement were weak or not detected (Figs. 2d, 5). These movements propagated caudally (4 dpf : 1.4 ± 0.1 cycles/min, 7 dpf : 1.7 ± 0.5 cycles/min, 8 dpf : 1.14 cycles/min v = 3.20 ± 1.22 μm/sec), which may correspond to the slow waves. They are distinct from peristaltic reflex or strong spontaneous 'peristaltic reflex-like' movement, either of which is associated with strong Ca 2+ events with slower cycles.
To investigate the function of the circular smooth muscles in gut movement, we expressed ChR2, a cation channel that is open upon irradiation with blue light 15 , by generating double transgenic fish SAGFF(LF)134A; Tg(UAS:ChR2-EYFP) 21 . Blue-light irradiation at a small spot caused a local constriction of the gut and closure of its lumen. We did not observe contraction at the anal or oral adjacent region ( Fig. 6a-g, Supplementary  videos 3 and 4). To investigate the functions of other types of cells in the gut, we generated double transgenic fish Tg(hsp70: Gal4); Tg(UAS: ChR2-EYFP). Heat shock at 6 dpf induced mosaic expression of ChR2 in some neurons at 8 dpf (Fig. 6h-q). Figure 6h-k shows an example in which a single neuron was found in a spot of blue light that extended axons in the oral direction (Supplementary video 5). In this case, contraction of the gut was observed at the oral side of the spot. The neuron may correspond to the descending interneuron ('3' in Fig. 1g). In Fig. 6l-q, two neurons expressing ChR2-EYFP were found in the ENS near the anus (Supplementary video 6). These neurons extend axons crossing the dorsal midline. When these neurons were irradiated with a spot of blue light, the rapid movement (7.7 ± 1.5 cycles/min, n = 3) of the anus was arrested immediately. They may represent inhibitory motoneurons or interneurons that regulate the inhibitory motoneurons. Heat shock at 3 dpf induced mosaic expression of ChR2-EYFP at 5 dpf in a different set of cells including mucosal epithelial cells and bowling-pin-shaped enterochromaffin (EC: cell '1' in Fig. 1f,g) or enteroendocrine cell-like cells. Irradiation at a small spot caused a local constriction of the gut and active movement at the oral part but not at the anal part, mimicking a peristaltic reflex-like movement (Fig. 6r-u, Supplementary video 7).
Our data represent the first example of Ca 2+ imaging of gut smooth muscles in zebrafish larvae in vivo as well as photo-manipulation of gut behavior, and they show evidence of a peristaltic reflex. The regular constriction of the gut at about 1 cycle/min may correspond to the slow waves produced by ICCs, and they may also correspond to the anterograde constrictions previously observed by video analysis in the larval gut without bolus 22 . Expression of markers for ICCs was reported in myenteric and submucosal layers in zebrafish 23 . These results provide a foundation on which to study the function of muscular systems, ENS, and other cell types in vivo in controlling gut behaviors at the single-cell level in the vertebrates.

Methods
Zebrafish. Zebrafish were raised and maintained according to standard procedures 24 and stage by day postfertilization at 28.5 °C (dpf) 25 . All procedures reported herein were carried out with the approval of the Institutional Animal Care and Use Committee of the University of Hyogo. All experiments were performed in accordance with the fundamental guidelines for proper conduct of animal experiment and related activities of the Ministry of Education, Culture, Sports, Science and Technology in Japan.
Tg(UAS:GCaMP3). The GCaMP3 gene was cloned into the EcoRI site of pUS 26 , giving rise to pUGCaMP3.
The DNA construct was injected with transposase mRNA into zebrafish embryos at the single-cell stage by the use of air pressure. The injected fish were screened by crossing with a Gal4 driver, Tg(dld: Gal4) 20 . Figure 6. Optogenetic activation of a small number of smooth muscle cells, neurons or endodermal cells could induce or arrest gut movement. (a-g) Activation of circular smooth muscles caused local contraction of the gut. Blue-light irradiation (indicated by a circle) of circular muscles expressing ChR2-eYFP in SAGFF(LF)134A; Tg(UAS: ChR2-eYFP) (a,b) and the control expressing GFP in SAGFF(LF)134A; Tg(UAS: GFP) (d,e) in the posterior gut at 8 dpf. The changes in the width of the gut (c,f). Green lines indicate the position of the measurement in the spot of blue light; red lines at the oral side of the spot; yellowgreen lines at the anal side. Blue lines in (c,f) indicate the periods when blue light was applied. Schematic representation of the gut movement induced by the spot light (g). Lateral views. Ant., anterior. Scale bars, 50 μm. Also see Supplementary videos 3 and 4. (h-k) Activation of a single neuron could induce contraction at the oral side of the neuron. Live view of the midgut (h) and the neuron expressing ChR2-eYFP in a spot of the blue light, extending one of the neurites anteriorly (i) and two kymographs of (g) at the oral-to-anal (green) and the dorsal-to-ventral (red) dashed lines (j). Schematic representation of the gut movement induced by the spot light (k). Scale bars, 50 μm for h and 10 μm for i. Also see Supplementary video 5. (l-q) Activation of two neurons located near the anus could cause the arrest of the local gut movement at 8 dpf of Tg(hsp70: Gal4); Tg(UAS: ChR2-eYFP). Live image of the gut (l). Ant., anterior. Double immunostaining for anti-GFP indicating the cells expressing ChR2-eYFP (green) and anti-5-HT (magenta) (m). Enlarged views of these neurons at lateral view (n) and dorsal view (o) indicating that some axons are extending across the dorsal midline of the gut. Dotted line indicates the dorsal midline. Kymograph at the dashed red line in (l). Schematic representation of the arrest of the gut movement induced by the spot of blue light (q). Scale bars, 20 μm. Also see Supplementary video 6. (r-u) Activation of cells in the endodermal cell layer could induce a peristaltic reflex-like movement (a local constriction of the gut and active movement at the oral part) at 5 dpf of Tg(hsp70: Gal4); Tg(UAS: ChR2-eYFP). The same gut before (r) and during (s) the irradiation with the spot of blue light. Kymographs at the solid and dashed red lines in (s, t). Schematic representation of the gut movement induced with the light (u). Asterisks indicate the lumen of the gut. Arrows indicate the movement of the gut. Scale bars, 20 μm. Also see Supplementary video 7.