Opposing effects of circadian clock genes bmal1 and period2 in regulation of VEGF-dependent angiogenesis in developing zebrafish

Molecular mechanisms underlying circadian-regulated physiological processes remain largely unknown. Here, we show that disruption of the circadian clock by both constant exposure to light and genetic manipulation of key genes in zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. Using a promoter-reporter system consisting of various deleted vegf-promoter mutants, we show that Bmal1 directly binds to and activates the vegf promoter via E-boxes. Additionally, we provide evidence that knockdown of Bmal1 leads to impaired Notch-inhibition-induced vascular sprouting. These results shed mechanistic insight on the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis.     

A central function for perlecan in skeletal muscle and cardiovascular development

Perlecan's developmental functions are difficult to dissect in placental animals because perlecan disruption is embryonic lethal. In contrast to mammals, cardiovascular function is not essential for early zebrafish development because the embryos obtain adequate oxygen by diffusion. In this study, we use targeted protein depletion coupled with protein-based rescue experiments to investigate the involvement of perlecan and its C-terminal domain V/endorepellin in zebrafish development. The perlecan morphants show a severe myopathy characterized by abnormal actin filament orientation and disorganized sarcomeres, suggesting an involvement of perlecan in myopathies. In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity. Live videomicroscopy confirms the abnormal swimming pattern caused by the myopathy and anomalous head and trunk vessel circulation. The phenotype is partially rescued by microinjection of human perlecan or endorepellin. These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities.     

Bmal1-deficient mouse fibroblast cells do not provide premature cellular senescence in vitro

Bmal1 is a core circadian clock gene. Bmal1^?/? mice show disruption of the clock and premature aging phenotypes with a short lifespan. However, little is known whether disruption of Bmal1 leads to premature aging at cellular level. Here, we established primary mouse embryonic fibroblast (MEF) cells derived from Bmal1^?/? mice and investigated its effects on cellular senescence. Unexpectedly, Bmal1^?/? primary MEFs that showed disrupted circadian oscillation underwent neither premature replicative nor stress-induced cellular senescence. Our results therefore uncover that Bmal1 is not required for in vitro cellular senescence, suggesting that circadian clock does not control in vitro cellular senescence.     

A requirement for <Emphasis Type="Italic">kit</Emphasis> in embryonic zebrafish melanocyte differentiation is revealed by melanoblast delay

Exploring differences in gene requirements between species can allow us to delineate basic developmental mechanisms, provide insight into patterns of evolution, and explain heterochronic differences in developmental processes. One example of differences in gene requirements between zebrafish and mammals is the requirement of the kit receptor tyrosine kinase in melanocyte development. kit is required for migration, survival and differentiation of all neural crest-derived melanocytes in mammals. In contrast, zebrafish kit is not required for differentiation of embryonic melanocytes during normal development. When melanoblast development in zebrafish embryos is delayed by injecting morpholinos targeted to the mitfa gene, we show that these delayed melanoblasts fail to differentiate in kit mutants. Thus, we show that there is a kit requirement for melanocyte differentiation in zebrafish when melanoblast development is delayed. Furthermore, we show that kit is not involved in maintaining melanocyte precursors through the developmental delay, but instead is required for differentiation of melanocytes after the block on their development is removed. Finally, we suggest there is a heterochronic shift in the onset of melanocyte differentiation between fish and mouse, and developmental delay of melanoblast development in zebrafish removes this heterochronic difference.Edited by D. Tautz     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

Betaglycan knock‐down causes embryonic angiogenesis defects in zebrafish

Angiogenesis is an essential requirement for embryonic development and adult homeostasis. Its deregulation is a key feature of numerous pathologies and many studies have shown that members of the transforming growth factor beta (TGF‐β) family of proteins play important roles in angiogenesis during development and disease. Betaglycan (BG), also known as TGF‐β receptor type III, is a TGF‐β coreceptor essential for mice embryonic development but its role in angiogenesis has not been described. We have cloned the cDNA encoding zebrafish BG, a TGF‐β‐binding membrane proteoglycan that showed a dynamic expression pattern in zebrafish embryos, including the notochord and cells adjacent to developing vessels. Injection of antisense morpholinos decreased BG protein levels and morphant embryos exhibited impaired angiogenesis that was rescued by coinjection with rat BG mRNA. In vivo time‐lapse microscopy revealed that BG deficiency differentially affected arterial and venous angiogenesis: morphants showed impaired pathfinding of intersegmental vessels migrating from dorsal aorta, while endothelial cells originating from the caudal vein displayed sprouting and migration defects. Our results reveal a new role for BG during embryonic angiogenesis in zebrafish, which has not been described in mammals and pose interesting questions about the molecular machinery regulating angiogenesis in different vertebrates. genesis 53:583–603, 2015. © 2015 Wiley Periodicals, Inc.     

Pluripotency in the light of the developmental hourglass

The hourglass model of development postulates divergence in early and late embryo development bridged by a period of developmental constraint at mid‐embryogenesis. Recently, molecular support for the hourglass model of development has accumulated, with the emphasis on studies using zebrafish and Drosophila species. Across mammals, the hourglass model and specifically divergence in early development has thus far received little attention. Divergence in mammalian pre‐implantation development is particularly interesting because of its potential impact on derivation of pluripotent embryonic stem cells. Here, we review recent findings that support the hourglass model of development. We provide striking examples of variation in key events in mammalian peri‐implantation development and their potential consequences for pluripotency of embryonic stem cell lines, including mechanisms of cell signalling and differentiation, gene regulatory networks, X‐chromosome inactivation, and epigenetic regulation. The variation in these processes indicates divergence in early mammalian development as was postulated by the hourglass model of development. We discuss the naive and primed states of pluripotency in light of this developmental divergence and their implications for human pluripotent stem cell states.     

Understanding dioxin developmental toxicity using the zebrafish model

Zebrafish (Danio rerio) have advantages over mammals as an animal model for investigating developmental toxicity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin, TCDD), a persistent global contaminant, is the most comprehensively studied developmental toxicant in zebrafish. The hallmark responses of TCDD developmental toxicity manifested in zebrafish larvae include edema, anemia, hemorrhage, and ischemia associated with arrested growth and development. Heart and vasculature development and function are severely impaired, and jaw malformations occur secondary to inhibited chondrogenesis. The swim bladder fails to inflate, and the switch from embryonic to adult erythropoiesis is blocked. This profile of developmental toxicity responses, commonly referred to as "blue sac syndrome" because the edematous yolk sac appears blue, is observed in the larval form of all freshwater fish species exposed to TCDD at the embryonic stage of development. Components of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AHR/ARNT) signaling pathway in zebrafish have been identified and functionally characterized. Their role in mediating TCDD toxicity has been determined using morpholinos to specifically knockdown the translation of zfAHR1, zfAHR2, zfARNT1, and zfARNT2 mRNAs, respectively, and a line of zfARNT2 null mutant zebrafish has provided further insight. These studies have shown that zfAHR2 and zfARNT1 mediate TCDD developmental toxicity. In addition, the growing use of molecular and genomic tools for research on zebrafish have led to advances in our understanding of the mechanism of TCDD developmental toxicity at the molecular level, including the recent finding that toxicity is not mediated by increased cytochrome P4501A (zfCYP1A) expression.     

Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes)

One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.     

Phylogenetic Analysis of Zebrafish Basic Helix-Loop-Helix Transcription Factors

The basic helix-loop-helix (bHLH) proteins play important regulatory roles in eukaryotic developmental processes including neurogenesis, myogenesis, hematopoiesis, sex determination, and gut development. Zebrafish is a good model organism for developmental biology. In this study, we identified 139 bHLH genes encoded in the zebrafish genome. Phylogenetic analyses revealed that zebrafish has 58, 29, 21, 5, 19, and 5 bHLH members in groups A, B, C, D, E, and F, respectively, while 2 members were classified as “orphan.” A comparison between zebrafish and human bHLH repertoires suggested that both organisms have a certain number of specific bHLH members. Eight zebrafish bHLH genes were found to have multiple coding regions in the genome. Two of these, Bmal1 and MITF, are good anchor genes for identification of fish-specific whole-genome duplication events in comparison with mouse and chicken genomes. The present study provides useful information for future studies on gene family evolution and vertebrate development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Impact of heart-specific disruption of the circadian clock on systemic glucose metabolism in mice

The daily rhythm of glucose metabolism is governed by the circadian clock, which consists of cell-autonomous clock machineries residing in nearly every tissue in the body. Disruption of these clock machineries either environmentally or genetically induces the dysregulation of glucose metabolism. Although the roles of clock machineries in the regulation of glucose metabolism have been uncovered in major metabolic tissues, such as the pancreas, liver, and skeletal muscle, it remains unknown whether clock function in non-major metabolic tissues also affects systemic glucose metabolism. Here, we tested the hypothesis that disruption of the clock machinery in the heart might also affect systemic glucose metabolism, because heart function is known to be associated with glucose tolerance. We examined glucose and insulin tolerance as well as heart phenotypes in mice with heart-specific deletion of Bmal1, a core clock gene. Bmal1 deletion in the heart not only decreased heart function but also led to systemic insulin resistance. Moreover, hyperglycemia was induced with age. Furthermore, heart-specific Bmal1-deficient mice exhibited decreased insulin-induced phosphorylation of Akt in the liver, thus indicating that Bmal1 deletion in the heart causes hepatic insulin resistance. Our findings revealed an unexpected effect of the function of clock machinery in a non-major metabolic tissue, the heart, on systemic glucose metabolism in mammals.     

Identification and expression analysis of mical family genes in zebrafish

Mical(molecule interacting with CasL)represent a conserved family of cytosolic multidomain proteins that has been shown to be associated with a variety of cellular processes,including axon guidance,cell movement,cell-cell junction formation,vesicle trafficking and cancer cell metastasis.However,the expression and function of these genes during embryonic development have not been comprehensively characterized,especially in vertebrate species,although some limited in vivo studies have been carried out in neural and musculature systems of Drosophila and in neural systems of vertebrates.So far,no mica/family homologs have been reported in zebrafish,an ideal vertebrate model for the study of developmental processes.Here we report eight homologs of m/ca/family genes in zebrafish and their expression profiles during embryonic development.Consistent with the findings in Drosophila and mammals,most zebrafish mical family genes display expression in neural and musculature systems.In addition,five mica/homologs are detected in heart,and one,micall2a,in blood vessels.Our data established an important basis for further functional studies of mica/family genes in zebrafish,and suggest a possible role for mica/genes in cardiovascular development.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Clock gene Bmal1 in mice embryo is dispensable for early embryo development but critical for live birth

In adult animals, the significance of circadian clocks in the regulation of physiology is well established. However, the physiological roles of embryonic clock genes on early embryo development, implantation and perinatal survival are still unclear. In the present study, using genotyping, embryo culture and transfer, the early embryo development, implantation, and perinatal survival of Bmal1+/+, Bmal1+/? and Bmal1?/? embryo were studied. At cleavage stage, the genotype ratio of Bmal1+/+, Bmal1+/? and Bmal1?/? embryo was 1:1.97:0.95, respectively (p > 0.05). Morula or early blastocyst developmental ratio was 83.8 ± 14.3, 87.1 ± 9.2 and 88.7 ± 14.5%, respectively (p > 0.05). After transferring of the three types of embryos to pseudopregnant wild-type mice, the implantation sites 4 days later was 7.7 ± 0.9, 7.2 ± 1.2 and 7.5 ± 0.5 (n = 4, F = 0.265, p = 0.773). Mean litter size of the mice after transferring with the three types of embryos was 5.5, 6.0, and 3.0 (n = 3, F = 30.3, p = 0.001). The development of Bmal1 null embryos was not impaired in preimplantation and early implantation stages, but the litter size had a trend to decrease.