Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2−/− knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFβ) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling—independent of Pitx2—controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis.

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Dendritic cells play a key role in processing and presenting antigens to naïve T cells to prime adaptive immunity. Circadian rhythms are known to regulate many aspects of immunity; however, the role of circadian rhythms in dendritic cell function is still unclear. Here, we show greater T cell responses when mice are immunised in the middle of their rest versus their active phase. We find a circadian rhythm in antigen processing that correlates with rhythms in both mitochondrial morphology and metabolism, dependent on the molecular clock gene, Bmal1. Using Mdivi-1, a compound that promotes mitochondrial fusion, we are able to rescue the circadian deficit in antigen processing and mechanistically link mitochondrial morphology and antigen processing. Furthermore, we find that circadian changes in mitochondrial Ca2+ are central to the circadian regulation of antigen processing. Our results indicate that rhythmic changes in mitochondrial calcium, which are associated with changes in mitochondrial morphology, regulate antigen processing.

Circadian rhythms broadly regulate physiological functions by tuning oscillations in the levels of mRNAs and proteins to the 24-hour day/night cycle. Globally assessing which mRNAs and proteins are timed by the clock necessitates accurate recognition of oscillations in RNA and protein data, particularly in large omics data sets. Tools that employ fixed-amplitude models have previously been used to positive effect. However, the recognition of amplitude-change in circadian oscillations required a new generation of analytical software to enhance the identification of these oscillations. To address this gap, we created the Pipeline for Amplitude Integration of Circadian Exploration (PAICE) suite. Here, we demonstrate the PAICE suite's increased detection of circadian trends through the joint modeling of the Mus musculus macrophage transcriptome and proteome. Our enhanced detection confirmed extensive circadian post-transcriptional regulation in macrophages, but highlighted that some of the reported discrepancy between mRNA and protein oscillations was due to noise in data. We further applied the PAICE suite to investigate the circadian timing of non-coding RNAs, documenting extensive circadian timing of long non-coding RNAs and small nuclear RNAs, which control the recognition of mRNA in the spliceosome complex. By tracking oscillating spliceosome complex proteins using the PAICE suite, we noted that the clock broadly regulates the spliceosome, particularly the major spliceosome complex. As most of the above-noted rhythms had damped amplitude changes in their oscillations, this work highlights the importance of the PAICE suite in the thorough enumeration of oscillations in omics-scale datasets.

Circadian rhythms broadly regulate physiological functions by tuning oscillations in the levels of mRNAs and proteins to the 24-hour day/night cycle. Globally assessing which mRNAs and proteins are timed by the clock necessitates accurate recognition of oscillations in RNA and protein data, particularly in large omics data sets. Tools that employ fixed-amplitude models have previously been used to positive effect. However, the recognition of amplitude-change in circadian oscillations required a new generation of analytical software to enhance the identification of these oscillations. To address this gap, we created the Pipeline for Amplitude Integration of Circadian Exploration (PAICE) suite. Here, we demonstrate the PAICE suite’s increased utility to detect circadian trends through the joint modeling of the Mus musculus macrophage transcriptome and proteome. Our enhanced detection confirmed extensive circadian post-transcriptional regulation in macrophages, but highlighted that some of the reported discrepancy between mRNA and protein oscillations was due to noise in data. We further applied the PAICE suite to investigate the circadian timing of non-coding RNAs, documenting extensive circadian timing of long non-coding RNAs and small nuclear RNAs, which control the recognition of mRNA in the spliceosome complex. By tracking oscillating spliceosome complex proteins using the PAICE suite, we noted that the clock broadly regulates the spliceosome, particularly the major spliceosome complex. As most of the above-noted rhythms had damped amplitude changes in their oscillations, this work highlights the importance of the PAICE suite in the thorough enumeration of oscillations in omics-scale datasets.

IntroductionSustained neuroinflammation is a major contributor to the progression of neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s (PD) diseases. Neuroinflammation, like other cellular processes, is affected by the circadian clock. Microglia, the resident immune cells in the brain, act as major contributors to neuroinflammation and are under the influence of the circadian clock. Microglial responses such as activation, recruitment, and cytokine expression are rhythmic in their response to various stimuli. While the link between circadian rhythms and neuroinflammation is clear, significant gaps remain in our understanding of this complex relationship. To gain a greater understanding of this relationship, the interaction between the microglial circadian clock and the enzyme NADPH Oxidase Isoform 2 (NOX2) was studied; NOX2 is essential for the production of reactive oxygen species (ROS) in oxidative stress, an integral characteristic of neuroinflammation.MethodsBV2 microglia were examined over circadian time, demonstrating oscillations of the clock genes Per2 and Bmal1 and the NOX2 subunits gp91phox and p47phox.ResultsThe BV2 microglial clock exerted significant control over NOX2 expression and inhibition of NOX2 enabled the microglia to retain a functional circadian clock while reducing levels of ROS and inflammatory cytokines. These trends were mirrored in mouse bone marrow-derived primary macrophages.ConclusionsNOX2 plays a crucial role in the interaction between the circadian clock and the activation of microglia/macrophages into their pro-inflammatory state, which has important implications in the control of neuroinflammation.

Sustained neuroinflammation is a major contributor to the progression of neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD) diseases. Neuroinflammation, like other cellular processes, is affected by the circadian clock. Microglia, the resident immune cells in the brain, act as major contributors to neuroinflammation and are under the influence of the circadian clock. Microglial responses such as activation, recruitment, and cytokine expression are rhythmic in their response to various stimuli. While the link between circadian rhythms and neuroinflammation is clear, significant gaps remain in our understanding of this complex relationship. To further our understanding of this relationship, we studied the interaction between the microglial circadian clock and the enzyme NADPH Oxidase Isoform 2 (NOX2), an enzyme essential for the production of reactive oxygen species (ROS) in oxidative stress, an integral characteristic of neuroinflammation. We examined BV2 microglia over circadian time, demonstrating oscillations of the clock genes Per2 and Bmal1 and the NOX2 subunits gp91phox and p47phox. We discovered the BV2 microglial clock exerted significant control over NOX2 expression and that the inhibition of NOX2 enabled the microglia to retain a functional circadian clock while reducing levels of ROS and inflammatory cytokines. These trends were mirrored in mouse bone marrow-derived primary macrophages. Our findings indicate NOX2 plays a crucial role in the interaction between the circadian clock and the activation of microglia/macrophages into their pro-inflammatory state.