Ca²⁺ dynamics in zebrafish morphogenesis

Zebrafish and Ca²⁺ imaging

Experiments were conducted as previously described (Tsuruwaka et al., 2007; Tsuruwaka, Konishi & Shimada, 2015). Briefly, 3 nL of synthetic YC 2.12 mRNA (0.5 ng/mL) was injected into blastodiscs of each single-cell embryo. After YC2.12 had confirmed to be distributed ubiquitously in the whole embryo, FRET analyses were performed as followed. Fluorescence images were obtained using a Zeiss Axiovert 200 microscope equipped with a combination of two filters, i.e., CFP-CFP, YFP-YFP, and CFP-YFP filters (Carl Zeiss, Oberkochen, Germany). Amplification and numerical aperture of the objective lens were 5× and 0.16, respectively. An AxioCam MRc5 camera (Carl Zeiss) was used to photograph the images, and the image analysis was performed using Axiovert FRET version 4.4 software (Carl Zeiss). Fluorescence was quantified following the manufacturer’s instructions. The control experiment was performed using Ca²⁺-ATPase inhibitor thapsigargin (Wako Pure Chemical Industries, Osaka, Japan) to confirm YC2.12 would work correctly (Schneider et al., 2008; Popgeorgiev et al., 2011). The number of eggs analyzed was 300 each experiment and the experiments were performed for total 37 times. Of those, 50 eggs were employed in the control experiment. No approval was required to conduct studies on fish according to the Ministry of Education, Culture, Sports, Science and Technology, Notice No. 71 (in effect since June 1, 2006).

Ca²⁺ dynamics during zebrafish morphogenesis

Ca²⁺ patterns showed dynamic changes during zebrafish morphogenesis (Fig. 1). Since the Ca²⁺ monitoring had been well studied with aquorin by Créton, Speksnijder & Jaffe (1998), we mainly focused on novel findings here. High Ca²⁺ levels were observed in the anterior and posterior body regions from stages bud to 16-somite (10–17 hpf). In the anterior trunk, the Ca²⁺ level reached a peak at 18-somite stage, whereas in the posterior trunk the Ca²⁺ peak was shown at 28-somite stage (Fig. S1).

In the developing head, the high level of Ca²⁺ was maintained through to prim-13 stage. Notably, this high Ca²⁺ level occurred concurrently with development of rhombomere, a segment of the developing hindbrain, from stages 26-somite to prim-10 (Fig. S2). Ca²⁺ level at presumptive midbrain increased at 26-somite stage and reached maximum level at prim-5 stage. Moreover, Ca²⁺ concentration at presumptive rhombomere 2 and 4 in hindbrain started to rise from 26-somite stage and then all rhombomeres showed relatively high Ca²⁺ levels at prim-5 stage. Ca²⁺ at rhombomere 2 reached maximum level at prim-5 stage, whereas rhombomere 1, 3 and 4 did at prim-6. With focusing on the rhombomere and midbrain hindbrain boundary (MHB), it is quite interesting to consider relevance between Ca²⁺ signals and formation of neuronal network. Ca²⁺ involves with neural network in zebrafish and Ca²⁺ sensors were used for studying neuronal activity and reflexive behavior (Higashijima et al., 2003; Muto et al., 2013; Portugues et al., 2014). Serial neural circuits such as sensory neuron, intercalated neuron, motor neuron, muscle were formed within 24 hpf in zebrafish (Saint-Amant & Drapeau, 1998; Downes & Granato, 2006; Fetcho, Higashijima & McLean, 2008; Pietri et al., 2009). When those circuits become active, zebrafish acquires stimulus-response. High Ca²⁺ levels at trunk and rhombomere regions in our results coincide with the development and activation of those circuits. Especially, Mauthner cells at rhombomere 4 become active and stimulate neural circuits, which results in triggering various body movements such as escape behavior (Korn & Faber, 2005). In fact, rhombomere and MHB during brain organization closely involved with Wnt signaling pathway which controls Ca²⁺ signaling (Webb & Miller, 2000; Prakash & Wurst, 2006). Therefore, Ca²⁺ dynamics at developing head in our results suggested intimate correlation with and formation and activation of neural circuits.

In the developing tail, the Ca²⁺ level had dropped by 20-somite stage and stabilized at a low level. The patterns in Ca²⁺ levels through the late gastrula and segmentation periods (Bud-28-somite stages; 10–23 hpf) that we obtained with yellow cameleon, YC2.12, were similar to those obtained previously with aequorin (Créton, Speksnijder & Jaffe, 1998; Webb & Miller, 2000). However, we succeeded in observing Ca²⁺patterns during early pharyngula period (Prim-5-20; 24–33 hpf) which have not reported anywhere yet.

Correlations between zebrafish morphogenesis and intracellular Ca²⁺ dynamics in the late gastrula-segmentation periods have been well characterized by Webb, Miller and colleagues (Gilland et al., 1999; Webb & Miller, 2007). Their work on Ca²⁺ dynamics during somitogenesis is particularly informative (Webb & Miller, 2010; Cheung et al., 2011; Webb et al., 2012).

Our finding of increasing Ca²⁺ levels in the anterior region during the pharyngula period, when the basic body plan is complete, is consistent with Ca²⁺-related gene expression, which controls the formation of the brain and nervous system (Zhou et al., 2008; Hsu & Tseng, 2010). Moreover, patterns of CaMK-II gene expression are in agreement with our observations of Ca²⁺ patterns at 3-somite, 18-somite, prim-5 stages and later, suggesting that this gene is closely involved with Ca²⁺ dynamics (Rothschild, Lister & Tombes, 2007). Figure S3 showed the compared images between our results and the CaMK-II expressions based on Rothschild, Lister & Tombes (2007). In fact, Freisinger et al. (2008) discuss correlations between Ca²⁺ signaling pathways and zebrafish body plan formation. The present study showed that cameleon, a genetically encoded Ca²⁺ sensor, enables us to analyze Ca²⁺ dynamics clearly during development and differentiation in a zebrafish embryo. YC2.12 worked correctly as Ca²⁺ sensor in whole living embryos since treatment with Ca²⁺-ATPase inhibitor thapsigargin induced altered Ca²⁺ level (Fig. S4). The embryo shown in Fig. S4B exhibited the increased Ca²⁺ level at later stages, which was consistent with the results reported by Popgeorgiev et al. (2011) (data not shown). We have achieved in tracking the serial Ca²⁺patterns from late gastrula to early pharyngula periods for the first time. This use of a variety of Ca²⁺ sensors has led to a novel perspective in the study of Ca²⁺ dynamics.

In future, tracking whole body Ca²⁺ signaling patterns with cameleon in addition to aequorin and other sensors may provide even more detail on Ca²⁺ signaling during zebrafish development. Thus, instead of discussing whether some Ca²⁺ indicators are superior to others, we propose that the use of a variety of indicators may give better results. Further comparison of our cameleon study results with those of previous Ca²⁺ studies should lead to more insight into Ca²⁺ dynamics.