Sarah McLoughlin

Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival

Postnatal knockout of a core clock gene in mice prompts reevaluation of the systemic role of the molecular clock in the biology of aging. For clock ticking, timing matters Ironically, antiaging product advertisements often promise to “slow down the clock.” But abolishing the circadian clock—for example, by knocking out Bmal1, a core clock gene—accelerates aging and shortens the life span in mice. As a result, Bmal1 knockout mice often serve as a model system in studies of the role of circadian rhythms in the aging process. Now Yang et al. show that the developmental timing of Bmal1 expression influences the circadian clock’s effects on aging and survival. To assess the role of circadian rhythms in the aging process, the authors made conditional Bmal1 knockout mice that are missing the BMAL1 protein only during adult life. Unlike knockout mice that perpetually lack Bmal1 expression, the new conditional Bmal1 knockout mice displayed loss of circadian rhythm in wheel-running activity, heart rate, and blood pressure, but exhibited normal life spans, fertility, body weight, blood glucose levels, and age-dependent arthropathy; in fact, atherosclerosis and hair growth actually improved, despite obliteration of clock function. Another surprising observation was little changes in overall gene expression in the livers of adult-life Bmal1 knockout mice, even though there’s a quelling of expression of oscillating genes. Both prenatal and postnatal knockout mice displayed similar ocular abnormalities and brain astrogliosis. Taken together, these findings reveal that many phenotypes thought to be caused by circadian rhythm disruption in conventional Bmal1 knockout mice apparently manifest as a result of clock-independent BMAL1 functions. Thus, the systemic role of the molecular clock in the biology of aging requires reinvestigation in order to increase the likelihood of translation for preclinical studies of the aging process. The absence of Bmal1, a core clock gene, results in a loss of circadian rhythms, an acceleration of aging, and a shortened life span in mice. To address the importance of circadian rhythms in the aging process, we generated conditional Bmal1 knockout mice that lacked the BMAL1 protein during adult life and found that wild-type circadian variations in wheel-running activity, heart rate, and blood pressure were abolished. Ocular abnormalities and brain astrogliosis were conserved irrespective of the timing of Bmal1 deletion. However, life span, fertility, body weight, blood glucose levels, and age-dependent arthropathy, which are altered in standard Bmal1 knockout mice, remained unaltered, whereas atherosclerosis and hair growth improved, in the conditional adult-life Bmal1 knockout mice, despite abolition of clock function. Hepatic RNA-Seq revealed that expression of oscillatory genes was dampened in the adult-life Bmal1 knockout mice, whereas overall gene expression was largely unchanged. Thus, many phenotypes in conventional Bmal1 knockout mice, hitherto attributed to disruption of circadian rhythms, reflect the loss of properties of BMAL1 that are independent of its role in the clock. These findings prompt reevaluation of the systemic consequences of disruption of the molecular clock.

Duplication and divergence of zebrafish CRALBP genes uncovers novel role for RPE- and müller-CRALBP in cone vision

PURPOSE During vertebrate phototransduction 11-cis-retinal is isomerized to all-trans-retinal. Light sensitivity is restored by recombination of apo-opsin with 11-cis-retinal to regenerate visual pigments. The conversion of all-trans retinal back to 11-cis-retinal is known as the visual cycle. Within the retina, cellular retinal-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Müller glia. CRALBP expressed in the RPE is known to facilitate the rate of the rod visual cycle. Recent evidence suggests a role for Müller glia in an alternate cone visual cycle. In this study, the role of RPE- and Müller-CRALBP in cone vision was characterized. METHODS The CRALBP orthologues rlbp1a and rlbp1b were identified in zebrafish by bioinformatic methods. The spatial and developmental expression of rlbp1a and rlbp1b was determined by in situ hybridization and immunohistochemistry. Depletion of the expression of the corresponding Cralbp a and Cralbp b proteins was achieved by microinjection of antisense morpholinos. Visual function was analyzed in 5-day post fertilization (dpf) larvae using the optokinetic response assay. RESULTS The zebrafish genome contains two CRALBP ohnologues, rlbp1a and rlbp1b. These genes have functionally diverged, exhibiting differential expression at 5 dpf in RPE and Müller glia, respectively. Depletion of CRALBP in the RPE or Müller glia results in abnormal cone visual behavior. CONCLUSIONS The results suggest that cone photoreceptors incorporate 11-cis-retinoids derived from the rod and cone visual cycles into their visual pigments and that Müller-CRALBP participates in the cone visual cycle.

Knitting Up the Raveled Sleave of Care

Recent advances in our understanding of molecular clocks highlight their relevance to human physiology and disease. Recent advances in our understanding of molecular clocks highlight their relevance to human physiology and disease. This Review is based on the Franklin Epstein Lecture delivered at Beth Israel Deaconess Hospital on 25 April 2013. We discuss recent advances in our understanding of molecular clocks and highlight their relevance to human physiology and disease.

Integrating multiple genome annotation databases improves the interpretation of microarray gene expression data

BackgroundThe Affymetrix GeneChip is a widely used gene expression profiling platform. Since the chips were originally designed, the genome databases and gene definitions have been considerably updated. Thus, more accurate interpretation of microarray data requires parallel updating of the specificity of GeneChip probes. We propose a new probe remapping protocol, using the zebrafish GeneChips as an example, by removing nonspecific probes, and grouping the probes into transcript level probe sets using an integrated zebrafish genome annotation. This genome annotation is based on combining transcript information from multiple databases. This new remapping protocol, especially the new genome annotation, is shown here to be an important factor in improving the interpretation of gene expression microarray data.ResultsTranscript data from the RefSeq, GenBank and Ensembl databases were downloaded from the UCSC genome browser, and integrated to generate a combined zebrafish genome annotation. Affymetrix probes were filtered and remapped according to the new annotation. The influence of transcript collection and gene definition methods was tested using two microarray data sets. Compared to remapping using a single database, this new remapping protocol results in up to 20% more probes being retained in the remapping, leading to approximately 1,000 more genes being detected. The differentially expressed gene lists are consequently increased by up to 30%. We are also able to detect up to three times more alternative splicing events. A small number of the bioinformatics predictions were confirmed using real-time PCR validation.ConclusionsBy combining gene definitions from multiple databases, it is possible to greatly increase the numbers of genes and splice variants that can be detected in microarray gene expression experiments.

Inhibition of the Pim1 Oncogene Results in Diminished Visual Function

Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3–5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors result in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function.

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

BackgroundGenetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue.ResultsHere, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype.ConclusionsWe conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

Clocks and Cardiovascular Function

Circadian clocks in central and peripheral tissues enable the temporal synchronization and organization of molecular and physiological processes of rhythmic animals, allowing optimum functioning of cells and organisms at the most appropriate time of day. Disruption of circadian rhythms, from external or internal forces, leads to widespread biological disruption and is postulated to underlie many human conditions, such as the incidence and timing of cardiovascular disease. Here, we describe in vivo and in vitro methodology relevant to studying the role of circadian rhythms in cardiovascular function and dysfunction.

Between a ROCK and a Hard Place How to Align Our Circadian Rhythms

Many, if not all, aspects of biology are temporally controlled to align our temperaments and behavior with a 24-hour world. Such rhythms are driven by the dynamic interplay of a master molecular clock, housed in the suprachiasmatic nucleus, and peripheral tissue clocks, all conditioned by environmental inputs, most of which we poorly understand.1 Expression of ≈20% of genes in most tissues oscillates in a circadian fashion,2 and the tightly regulated transcriptional regulation of the clock is complemented by additional controls—by microRNAs3 and posttranslational4 and epigenetic modifications.5 Further underscoring the importance of this system, the molecular clock is highly conserved, exhibits marked elemental redundancy, and is central among the networks that link biological networks that are prominent in distinct tissues.1 Article see p 104 Longstanding clinical interest in circadian rhythms has been fostered by temporal variability in the incidence of symptoms of many diseases1—asthma, endogenous depression, myocardial infarction, and stroke among them—as well as in the efficacy and metabolism of commonly used drugs.6 Coincident with the diurnal pattern of cardiovascular events, diurnal variations have been reported in the pressor response to infused vasoconstrictors, sympathetic nerve activity, the renin–angiotensin system, QT …