Regular heartbeat rhythm at the heartbeat initiation stage is essential for normal cardiogenesis at low temperature
- Tomomi Watanabe-Asaka†1,
- Yoshio Sekiya†1,
- Hironori Wada2,
- Takako Yasuda1,
- Ikuya Okubo1,
- Shoji Oda1 and
- Hiroshi Mitani1Email author
© Watanabe-Asaka et al.; licensee BioMed Central Ltd. 2014
Received: 12 November 2013
Accepted: 14 February 2014
Published: 25 February 2014
The development of blood flow in the heart is crucial for heart function and embryonic survival. Recent studies have revealed the importance of the extracellular matrix and the mechanical stress applied to the valve cushion that controls blood flow to the formation of the cardiac valve during embryogenesis. However, the events that trigger such valve formation and mechanical stress, and their temperature dependence have not been explained completely. Medaka (Oryzias latipes) inhabits a wide range of East Asia and adapts to a wide range of climates. We used medaka embryos from different genomic backgrounds and analyzed heartbeat characteristics including back-and-forth blood flow and bradyarrhythmia in embryos incubated at low temperature. We also used high-speed imaging analysis to examine the heartbeat of these animals after transient exposure to low temperature.
Embryos of the Hd-rR medaka strain exhibited back-and-forth blood flow in the heart (blood regurgitation) after incubation at 15°C. This regurgitation was induced by exposure to low temperature around the heartbeat initiation period and was related to abnormalities in the maintenance or pattern of contraction of the atrium or the atrioventricular canal. The Odate strain from the northern Japanese group exhibited normal blood flow after incubation at 15°C. High-speed time-lapse analysis of the heartbeat revealed that bradyarrhythmia occurred only in Hd-rR embryos incubated at 15°C. The coefficient of contraction, defined as the quotient of the length of the atrium at systole divided by its length at diastole, was not affected in either strain. The average heart rate after removing the effect of arrhythmia did not differ significantly between the two strains, suggesting that the mechanical stress of individual myocardial contractions and the total mechanical stress could be equivalent, regardless of the presence of arrhythmia or the heart rate. Test-cross experiments suggested that this circulation phenotype was caused by a single major genomic locus.
These results suggest that cardiogenesis at low temperature requires a constant heartbeat. Abnormal contraction rhythms at the stage of heartbeat initiation may cause regurgitation at later stages. From the evolutionary viewpoint, strains that exhibit normal cardiogenesis during development at low temperature inhabit northern environments.
KeywordsHeartbeat Medaka Blood regurgitation Cold adaptation Cardiogenesis
The heart is an organ that constantly circulates blood to the body, which is essential for animal survival. Congenital heart defects affect a few percent of human newborns each year [1, 2]. Defects in the heartbeat are categorized into two types: impaired control of blood flow caused by a deformed valve and impaired contraction caused by abnormal cardiomyocytes. A rhythmic heartbeat is important for supplying oxygen to the whole body via the blood; therefore, arrhythmia is a relatively severe heart disease. In particular, the cardiac valve is essential to determining unidirectional blood flow, and 20–30% of cardiac defects are caused by heart valve abnormalities . The recent literature has identified the key factors and pathways involved in valve development, such as extracellular matrix (ECM) formation and the importance of mechanical stress [3–6]. However, the mechanisms underlying the generation of the rhythmic contractions of the heartbeat during embryogenesis and the timing or events required for valve formation remain unknown.
Mutants with defects in heart formation have been isolated in medaka and zebrafish, some of which have heartbeat abnormalities [7–14]. The requirement of ECM components, such as chondroitin sulfate, for the formation of the atrioventricular canal (AVC) to prevent blood regurgitation during development of the heart has been shown in zebrafish embryos. The importance of mechanical stresses to valve formation was demonstrated by experiments that used microbeads to induce physical blockage and by mathematical models [4–7, 9, 15–17]. Temperature is one important factor that affects the growth of poikilothermic animals. Consequently, the acquisition of broad temperature adaptability enables expansion into a new environment. Temperature-sensitive mutants with abnormal phenotypes in heartbeat rhythm at the heartbeat initiation stage and blood flow regulation at the later stage, as detected using large-scale mutagenesis screening, have not been reported to date.
In this study, we identified the critical stage of normal heart development under low-temperature conditions using medaka embryos of an inbred strain derived from the “southern Japanese group” (S.JPN). High-speed image analysis revealed that the heartbeat rhythm at the stage of its onset was important for the establishment of normal blood circulation. Moreover, differences in heart development in embryos grown at cold temperature were observed between the S.JPN and the “northern Japanese group” (N.JPN). The genetic cross test used in this study showed that the cold-sensitive phenotype followed a Mendelian recessive inheritance.
Blood regurgitation in medaka embryos grown at low temperature
Additional file 2: Movie S1: Frontal view of an Hd-rR control embryo at st.34 (300 fps movie). The blood flow was unaffected in low-temperature-treated embryos. (MPEG 5 MB)
Additional file 3: Movie S2: Frontal view of an Hd-rR embryo with regurgitation at st.36 (300 fps movie). Blood flow was regurgitated in the heart. (MPEG 5 MB)
Average heart rate of st.34 medaka embryos measured at 26°C
Incubated at 15°C
Heart rate (bpm; mean ± SD)
116.0 ± 10.6
117.5 ± 1.82
Critical period for blood regurgitation at low temperature
Subsequently, to identify the period that was most critical for blood regurgitation, Hd-rR embryos were incubated at 15°C from st.18 to the nine-somite stage (st.22) or to st.24, as depicted in Figure 3B. Blood regurgitation at st.34 did not occur in embryos exposed to cold at st.18–st.22 (low temperature: L18–22), although a percentage of the embryos showed blood regurgitation significantly increased to 12.1% (P = 0.014) in the embryos exposed to cold at st.18–st.24 (L18–24) (Figure 3C). In medaka, st.22, st.23 and st.24 embryos usually reach to the next developmental stages for 3, 3 and 6 hours at 26.5°C . Next, the 15°C treatment was interrupted briefly by the 3-hour-incubation at 26.5°C from st.22 (breakage of 15°C treatment around st.22-23: B22–23), st.23 (B23–24) and st.24 (B24–25) to identify the stage that was critical for blood regurgitation. The frequency of the severe phenotype was significantly reduced in B22–23 and B23–24 embryos compare to the control embryos (Figure 2C; p = 0.019 and p = 0.0001, respectively). However, there was no rescue in the regurgitation phenotype by the break in the 15°C treatment at st.24–25. These results suggest that incubation at 15°C around the heartbeat initiation stage is sufficient to induce blood regurgitation in medaka and that this blood regurgitation phenotype is irreversible after st.34.
Differences in phenotypic variations in blood regurgitation between medaka groups
Comparative heartbeat analysis in st.24 and st.34 embryos of the Hd-rR and ODT strains
Average heartbeat measured at 15°C and 26°C in st.24 and st.34 medaka embryos of the Hd-rR and ODT strains
Heart rate (mean ± SD)
9.08 ± 1.66
24.0 ± 4.43
8.78 ± 2.67
21.1 ± 3.93
70.7 ± 3.21
116.0 ± 10.6*
67.3 ± 2.31
116.9 ± 6.44
To verify the possibility that a reduction in heart rate caused blood regurgitation, the average heart rate was calculated for 10 beats. Some ODT embryos exhibited a slower average heart rate than did Hd-rR embryos (Figure 5E). Average heart rate including arrhythmias in the Hd-rR and ODT embryos are 8.48 ± 2.74 and 5.74 ± 1.51, respectively (p <0.05). This result apparently seems that heart rate under low temperature is slower in the Hd-rR strain than the ODT. However the difference between these strains is mainly due to the bradyarrhythmia in the Hd-rR strain. After removing the bradyarrhythmias to compare the intrinsic heart rate under low temperature, the average heart rates did not differ between the Hd-rR and ODT strains: 9.08 ± 1.66 and 8.78 ± 2.67 bpm, respectively (Figure 5E and Table 2; p = 0.80). The heart rate was constant, and no arrhythmia was observed in either strain at 26°C (Table 2). These results suggest that incubation at 15°C during the early stage of development of the heartbeat disturbs the rhythm but not the heart rate in Hd-rR embryos.
Terfenadine is a K+-channel blocker that disturbs the heartbeat even in small teleosts [20, 21]. To examine whether K+-channel blockage could account for the observed rhythm phenotype of the heartbeat, terfenadine was added to Hd-rR and ODT embryos at st.24 and st.34. Atrioventricular blockage was induced in the terfenadine-treated embryos at the heart development stage (st.36) without blood regurgitation. By contrast, terfenadine did not affect the heartbeat at st.24 (data not shown).
Pattern of inheritance of the blood regurgitation and bradyarrhythmia phenotype
Frequency of braddyarrhythmia at 15°C in st.24 and st.34 medaka embryos of the Hd-rR and ODT strains
F1 × F1
F1 × Hd-rR
Mechanisms causing the irregular heartbeat observed at low temperature
Our results showed that low temperature caused bradyarrhythmia in Hd-rR embryos at the heartbeat initiation stage. In the chick embryo, this heartbeat initiation stage is highly sensitive to low temperature, and cooling causes disorder in the rhythmicity of the action potential . During embryogenesis, the heart is the only organ that has rhythmicity; therefore, the blood circulation would be especially sensitive to low temperature. It is thought that the Ca2+ channel functions as the pacemaker in the early embryonic heart and that it is important to maintain the Ca2+ signal in the cytoplasm . The results of the present study show that the K+ channel is irrelevant, implying that the stability of Ca2+ channels might be crucial at low temperatures. It is known that the rhythmicity of this Ca2+ concentration is mediated by several components such as hyperpolarization-activated cation channel 4; however, the interactions of these molecules are complicated.
Adaptation to low temperature in medaka wild populations
In their natural environment, medaka in Japan spawn from the end of April to August. It is known that embryos of medaka strains of the S.JPN group develop normally at 16–34°C and that blood stagnation or impaired blood vessel formation causes failure in hatching at temperatures below 16°C . However, at the beginning of spring, medaka embryos in the northern part of Japan frequently experience temporarily low temperatures below 16°C in the field [25–27]. Odate, which is located at high latitude, is a habitat of the N.JPN population and is the original location for the capture of fish of the ODT strain. The maximum and minimum temperatures in May (spawning period) are around 20°C and 10°C, respectively (http://www.data.jma.go.jp/obd/stats/etrn). About 100 wild populations of medaka have been maintained as closed colonies in an outdoor breeding farm at the University of Tokyo, Graduate School of Frontier Sciences, since 1985 . The maximum and minimum water temperatures at which the N.JPN group, including the ODT strain, starts spawning are around 18°C and 10°C, respectively.
Relationship between the disturbance of the heart rhythm and cardiac hypoplasia
The results in this study suggest that the regurgitation observed after incubation at low temperature was caused by a structural defect, such as in the function of the canals or the atrium, and not by a transient, morphological or neurogenic dysfunction. Three possible mechanisms may have caused the blood regurgitation phenotype observed: an unclosed valve that was not formed properly, an irregular rhythm, or abnormal atrial and ventricular contractions. Our results showed that the rhythm of the heartbeat and the coefficient of the contraction were not affected in the regurgitated embryos at st.34. There were no apparent morphological abnormalities; therefore, very subtle differences in the AVC structure may be important to continue proper heartbeat. Conversely, the presence of retrograde flow with or without valves in some zebrafish mutants has been reported [7, 9]. The endocardial cushion plays a role as a valve in the early stages of zebrafish and medaka development [12, 29, 30]. Cardiac physiologists have long conjectured that the valveless embryonic heart tube drives circulation via peristaltic or impedance contractions [5, 31–33].
It has been reported that the heart formation of medaka embryo of st.34 is similar to zebrafish embryos of stage 56 hours post fertilization (hpf)  and that the cardiac valve at 56 hpf is known as the cushion in zebrafish . The heartbeat could be distinguished in the period of the isovolumic ventricular contraction period (a in Figure 2E–G), the atrial contraction/ventricular dilatation period (b in Figure 2E–G), and the ventricular contraction and ejection period (c in Figure 2E–G). The results of this study show normal contractions in the ventricle (b and c in Figure 2E–G) and in the atrium (b in Figure 2E–G), and abnormalities in the maintenance of the contraction in the atrium and pattern of the contraction in the AVC. The diameter of the ventricle during period a in Figure 2E–G is smaller in the regurgitating embryo. It is possible that the ventricular diameter is reduced by the increased pressure in the pericardium caused by the relaxed atrium and AVC. This might explain the blood regurgitation observed in the Hd-rR strain.
The heart in medaka is positioned between the yolk and the embryo during embryogenesis, and this situation causes difficulties observing the outflow tract in medaka. However, it is less likely that the blood regurgitation is caused by the structure of the outflow tract because of the normal ventricular contraction in the period (c in Figure 2E–G). Hence, the low-temperature-induced regurgitation may reflect an abnormality in the pattern or in the maintenance of the contraction in the atrium or the AVC, especially in the endocardial cushion. The results showing that a constant heartbeat was necessary for normal development in the ODT strain at 15°C in the present study support the importance of sheer stress induced by the rhythmic contraction to the retrograde flow afterwards. Unidirectional rhythmic contraction is required for ATP release and Ca2+ signaling in blood vessels and microRNA expression in the valve appear with the heartbeat [34, 35]. A similar mechanism could also play an important role in the initial stages of heart formation. Direct evidence about whether a structural defect exists is needed to confirm this hypothesis in the future.
In conclusion, the low-temperature-induced disturbance of the rhythm of the heartbeat in these embryos was associated with irreversible blood regurgitation observed in the AVC during embryonic development at later stages. Intercrossing experiments suggested the existence of a single major gene related to this phenotype. The identification of the genetic locus that contributes to cold sensitivity in medaka would represent another breakthrough in the study of cold adaptation and of its relationship with the population diversity of this species.
Fish maintenance and husbandry
The medaka (Oryzias latipes) inbred strain Hd-rR (derived from the S.JPN group) was obtained from our breeding colony. We also used local strains (demes): ODT, KAM, KAG, and MIY strains from the N.JPN group; KES, IID, and KAS strains from the S.JPN group; and SYA strain from a western Korean/Chinese group. All animals in this study were kept as closed colonies under laboratory conditions. Eggs were collected and incubated in Petri dishes (φ 60 mm) with tap water containing 0.0001% methylene blue. The designation of medaka developmental stages was in accordance with that of Iwamatsu (2004) . The fish were maintained under standard laboratory conditions at 26–29°C with a 14 h:10 h light:dark photoperiod and were fed three times a day with live brine shrimp and commercial pellet food. No significant differences in whole-body development were observed between strains. The Committees for Institutional Animal Care of the University of Tokyo approved the animal protocols.
Low-temperature treatment and observation of the blood circulation
The fertilized eggs were incubated at ambient temperature (~26.5°C) until the developmental stage needed for the experiments. In each experimental condition, more than 30 embryos were exposed to a temperature of 15°C at certain periods of development. The egg water was changed once a week. The contraction of the heart at st.24 was calculated after a 4 h incubation at 15°C. In the control medaka, there was some retrograde blood flow until st.33, the stage at which notochord vacuoliazation is completed, and chorions became turbid and fragile at st.39 before hatching. Heartbeat and blood circulation were observed at st.24, st.34, and st.38. Blood flow was classified into three patterns based on the level of the back flow to the atrium: the absence of back flow or the return of a few dozen blood cells into the atrium was classified as “normal”; the presence of blood regurgitation but with continuing circulation was classified as “moderate”; and the cessation of circulation because of blood regurgitation into the heart was classified as “severe”.
Digital video recording, extraction of images showing heart movement, and heartbeat analysis
Embryos in the chorion were immobilized in the correct orientation using 2.5% methylcellulose in glass-bottom dishes. Terfenadine (final concentration 10 μM in 0.1% DMSO) was added 2 h before video recording. Retrograde blood flow from the ventricle to the atrium was assessed at 15°C or 26°C using a stereomicroscope (Nikon MULTIZOOM AZ100) equipped with a digital high-speed camera (CASIO Exilim EX-F1) . Digital pictures were captured at 30 or 300 frames/s (fps) at a resolution of 512 × 384 pixels for up to 3 min and recorded in a PC using Final Cut Pro software (Apple Inc., Cupertino, USA).
Movies of heart movements were processed using Bohboh software (BohbohSoft, Tokyo, Japan). Blood flow and contraction rhythms were measured based on alterations in the intensity of blood flow into and out of the atrium. Regions of interest (ROIs) in the atrium were selected. The pixel intensities of the ROIs were digitized throughout the entire time series examined using Bohboh and were processed further using Cutwin mathematical software (EverGreen Soft, Tokyo, Japan). The data were processed using a smoothing algorithm by taking the movement average over 21 frames to correct for peak-detection errors. Average heart rates were obtained from the number of maxima recorded in 1 min. The time between maxima gave a measure of the interbeat interval. Bradyarrhythmia was identified as the interbeat interval whose duration was more than twice that of the minimal interbeat interval. Bradyarrhythmias were excluded from the coefficient of variance and heart rate analyses. Heart rate variation was obtained as the difference of each interbeat interval from the average value.
The external diameter of the ventricle, atrium, and AVC were selected. The length of each region was digitized throughout the entire time series examined using Bohboh and processed further using Cutwin. The data were processed using a smoothing algorithm by taking the movement average over 21 frames to correct for peak-detection errors. Two heartbeats were plotted as typical heartbeats. The coefficient of the contraction was calculated as the quotient of the length of the atrium at systole divided by its length at diastole. Calculations were performed three times in each embryo.
Statistical analyses were performed using Fisher’s exact test and one-way ANOVA using JMP software (SAS Japan, Tokyo, Japan). A p value < 0.05 was considered significant. Data are presented as the mean ± SD of the indicated samples per experiment.
Frames per second
The Northern Japanese group
Region of interest
The Southern Japanese group
We express our appreciation to Mmes. Shizuko Chiba and Sumiko Tomizuka for maintaining of medaka strains. This work was supported by Grants-in-Aid for Scientific Research (21221003 to HM, 25860177 to T-WA and 24310039 to SO) from the Ministry of Education, Sports, Science and Technology (MEXT) of Japan.
- Robinson A LMG: Clinical Genetic Handbook. 1993, Boston: Blackwell Scientific PublicationsGoogle Scholar
- Hoffman JI, Kaplan S: The incidence of congenital heart disease. J Am Coll Cardiol. 2002, 39: 1890-1900. 10.1016/S0735-1097(02)01886-7.View ArticleGoogle Scholar
- Puceat M: Embryological origin of the endocardium and derived valve progenitor cells: from developmental biology to stem cell-based valve repair. Biochim Biophys Acta. 1833, 2013: 917-922.Google Scholar
- Staudt D, Stainier D: Uncovering the molecular and cellular mechanisms of heart development using the zebrafish. Annu Rev Genet. 2012, 46: 397-418. 10.1146/annurev-genet-110711-155646.View ArticleGoogle Scholar
- Santhanakrishnan A, Miller LA: Fluid dynamics of heart development. Cell Biochem Biophys. 2011, 61: 1-22. 10.1007/s12013-011-9158-8.View ArticleGoogle Scholar
- Miller LA: Fluid dynamics of ventricular filling in the embryonic heart. Cell Biochem Biophys. 2011, 61: 33-45. 10.1007/s12013-011-9157-9.View ArticleGoogle Scholar
- Bartman T, Walsh EC, Wen KK, McKane M, Ren J, Alexander J, Rubenstein PA, Stainier DY: Early myocardial function affects endocardial cushion development in zebrafish. PLoS Biol. 2004, 2: E129-10.1371/journal.pbio.0020129.View ArticleGoogle Scholar
- Walsh EC, Stainier DY: UDP-glucose dehydrogenase required for cardiac valve formation in zebrafish. Science. 2001, 293: 1670-1673. 10.1126/science.293.5535.1670.View ArticleGoogle Scholar
- Stainier DY, Fouquet B, Chen JN, Warren KS, Weinstein BM, Meiler SE, Mohideen MA, Neuhauss SC, Solnica-Krezel L, Schier AF, Zwartkruis F, Stemple DL, Malicki J, Driever W, Fishman MC: Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development. 1996, 123: 285-292.Google Scholar
- Chen JN, Haffter P, Odenthal J, Vogelsang E, Brand M, van Eeden FJ, Furutani-Seiki M, Granato M, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh RN, Mullins MC, Nusslein-Volhard C: Mutations affecting the cardiovascular system and other internal organs in zebrafish. Development. 1996, 123: 293-302.Google Scholar
- Fujita M, Komeda M, Hasegawa K, Kihara Y, Nohara R, Sasayama S: Pericardial fluid as a new material for clinical heart research. Int J Cardiol. 2001, 77: 113-118. 10.1016/S0167-5273(00)00462-9.View ArticleGoogle Scholar
- Taneda Y, Konno S, Makino S, Morioka M, Fukuda K, Imai Y, Kudo A, Kawakami A: Epigenetic control of cardiomyocyte production in response to a stress during the medaka heart development. Dev Biol. 2010, 340: 30-40. 10.1016/j.ydbio.2010.01.014.View ArticleGoogle Scholar
- Glenn NO, McKane M, Kohli V, Wen KK, Rubenstein PA, Bartman T, Sumanas S: W-loop of alpha-cardiac actin is critical for heart function and endocardial cushion morphogenesis in zebrafish. Mol Cell Biol. 2012, 32: 3527-3540. 10.1128/MCB.00486-12.View ArticleGoogle Scholar
- Zhao L, Zhao X, Tian T, Lu Q, Skrbo-Larssen N, Wu D, Kuang Z, Zheng X, Han Y, Yang S, Zhang C, Meng A: Heart-specific isoform of tropomyosin4 is essential for heartbeat in zebrafish embryos. Cardiovasc Res. 2008, 80: 200-208. 10.1093/cvr/cvn177.View ArticleGoogle Scholar
- Peal DS, Burns CG, Macrae CA, Milan D: Chondroitin sulfate expression is required for cardiac atrioventricular canal formation. Dev Dyn. 2009, 238: 3103-3110. 10.1002/dvdy.22154.View ArticleGoogle Scholar
- Armstrong EJ, Bischoff J: Heart valve development: endothelial cell signaling and differentiation. Circ Res. 2004, 95: 459-470. 10.1161/01.RES.0000141146.95728.da.View ArticleGoogle Scholar
- Hove JR, Koster RW, Forouhar AS, Acevedo-Bolton G, Fraser SE, Gharib M: Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature. 2003, 421: 172-177. 10.1038/nature01282.View ArticleGoogle Scholar
- Fujita M, Mitsuhashi H, Isogai S, Nakata T, Kawakami A, Nonaka I, Noguchi S, Hayashi YK, Nishino I, Kudo A: Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro. Dev Biol. 2012, 361: 79-89. 10.1016/j.ydbio.2011.10.008.View ArticleGoogle Scholar
- Iwamatsu T: Stages of normal development in the medaka Oryzias latipes. Mech Dev. 2004, 121: 605-618. 10.1016/j.mod.2004.03.012.View ArticleGoogle Scholar
- Dhillon SS, Doro E, Magyary I, Egginton S, Sik A, Muller F: Optimisation of embryonic and larval ECG measurement in zebrafish for quantifying the effect of QT prolonging drugs. PLoS One. 2013, 8: e60552-10.1371/journal.pone.0060552.View ArticleGoogle Scholar
- Chan PK, Lin CC, Cheng SH: Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos. BMC Biotechnol. 2009, 9: 11-10.1186/1472-6750-9-11.View ArticleGoogle Scholar
- Hirota A, Fujii S, Sakai T, Kamino K: Temperature dependence of spontaneous electrical activity in early embryonic heart monitored optically with a potential-sensitive dye. Jpn J Physiol. 1983, 33: 85-100. 10.2170/jjphysiol.33.85.View ArticleGoogle Scholar
- Sakai T, Fujii S, Hirota A, Kamino K: Optical evidence for calcium-action potentials in early embryonic precontractile chick heart using a potential-sensitive dye. J Membra Biol. 1983, 72: 205-212. 10.1007/BF01870587.View ArticleGoogle Scholar
- Hirayama M, Mukai T, Miya M, Murata Y, Sekiya Y, Yamashita T, Nishida M, Watabe S, Oda S, Mitani H: Intraspecific variation in the mitochondrial genome among local populations of Medaka Oryzias latipes. Gene. 2010, 457: 13-24. 10.1016/j.gene.2010.02.012.View ArticleGoogle Scholar
- Iwamatsu T: The Integrated Book for the Biology of the Medaka. 1997, Okayama: University Education PressGoogle Scholar
- Tsukuda H, Katayama T: Temperature adaptation in fish I. Influences of incubation temperature on temperature tolerence, growth and physical formation. Physiol Ecol. 1957, 7: 113-122.Google Scholar
- Hirayama M, Mitani H, Watabe S: Temperature-dependent growth rates and gene expression patterns of various medaka Oryzias latipes cell lines derived from different populations. J Comp Physiol B. 2006, 176: 311-320. 10.1007/s00360-005-0053-8.View ArticleGoogle Scholar
- Mitani H, Kamei Y, Fukamachi S, Oda S, Sasaki T, Asakawa S, Todo T, Shimizu N: The medaka genome: why we need multiple fish models in vertebrate functional genomics. Genome Dyn. 2006, 2: 165-182.View ArticleGoogle Scholar
- Scherz PJ, Huisken J, Sahai-Hernandez P, Stainier DY: High-speed imaging of developing heart valves reveals interplay of morphogenesis and function. Development. 2008, 135: 1179-1187. 10.1242/dev.010694.View ArticleGoogle Scholar
- Combs MD, Yutzey KE: Heart valve development: regulatory networks in development and disease. Circ Res. 2009, 105: 408-421. 10.1161/CIRCRESAHA.109.201566.View ArticleGoogle Scholar
- Forouhar AS, Liebling M, Hickerson A, Nasiraei-Moghaddam A, Tsai HJ, Hove JR, Fraser SE, Dickinson ME, Gharib M: The embryonic vertebrate heart tube is a dynamic suction pump. Science. 2006, 312: 751-753. 10.1126/science.1123775.View ArticleGoogle Scholar
- Manner J, Wessel A, Yelbuz TM: How does the tubular embryonic heart work? Looking for the physical mechanism generating unidirectional blood flow in the valveless embryonic heart tube. Dev Dyn. 2010, 239: 1035-1046. 10.1002/dvdy.22265.View ArticleGoogle Scholar
- Hickerson AI, Rinderknecht D, Gharib M: Experimental study of the behavior of a valveless impedance pump. Exp Fluids. 2005, 38: 534-540. 10.1007/s00348-005-0946-z.View ArticleGoogle Scholar
- Banjo T, Grajcarek J, Yoshino D, Osada H, Miyasaka KY, Kida YS, Ueki Y, Nagayama K, Kawakami K, Matsumoto T, Sato M, Ogura T: Haemodynamically dependent valvulogenesis of zebrafish heart is mediated by flow-dependent expression of miR-21. Nat Commun. 1978, 2013: 4-Google Scholar
- Yamamoto K, Furuya K, Nakamura M, Kobatake E, Sokabe M, Ando J: Visualization of flow-induced ATP release and triggering of Ca2+ waves at caveolae in vascular endothelial cells. J Cell Sci. 2011, 124: 3477-3483. 10.1242/jcs.087221.View ArticleGoogle Scholar
- Watanabe-Asaka T, Niihori M, Terada M, Oda S, Iwasaki K, Sudoh M, Yamada S, Ohshima H, Mukai C: Technologies to analyze the movement of internal organs by high-speed movies using medaka. Transac Jpn Soc Aeronautica Space Sci Aerospace Technol Jpn. 2012, 10: Pp_1-Pp_4. 10.2322/tastj.10.1.View ArticleGoogle Scholar
- Wada H, Ghysen A, Satou C, Higashijima S, Kawakami K, Hamaguchi S, Sakaizumi M: Dermal morphogenesis controls lateral line patterning during postembryonic development of teleost fish. Dev Biol. 2010, 340: 583-594. 10.1016/j.ydbio.2010.02.017.View ArticleGoogle Scholar
- Yasuda T, Oda S, Li Z, Kimori Y, Kamei Y, Ishikawa T, Todo T, Mitani H: Gamma-ray irradiation promotes premature meiosis of spontaneously differentiating testis-ova in the testis of p53-deficient medaka (Oryzias latipes). Cell Death Dis. 2012, 3: e395-10.1038/cddis.2012.133.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.