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Genes encoding C2H2-type zinc finger (ZF) proteins constitute one of the largest gene families in animal genomes. Many C2H2-type ZF domains are used for DNA-binding, and others are used for protein–protein interactions and RNA-binding. In ascidian embryos, a gene regulatory network controlling cell fate decision has been studied extensively. However, because ZF proteins have not been well annotated genome-widely, it has not been clear which ZF proteins are possible regulators for cell fate decision in this animal. Therefore, we tried to identify and classify all genes encoding C2H2-type ZF proteins in the genome of an ascidian in the present study. Because the number of ZF domains often varies among possible orthologous proteins, and because ZF domains are not long enough for molecular phylogenetic analyses in many cases, we extensively utilized other bioinformatics tools for their classification. In addition, we found that many ascidian ZF proteins contain fewer ZF domains than their human orthologs; this finding might be related to the compactness of the ascidian genome.

Plexins are axon guidance transmembrane receptors that control cytoskeleton and membrane dynamics in development and adult physiology. As plexins are expressed in multiple cell types in various tissues, floxed alleles that enable conditional deletion are needed to facilitate cell type-specific functional analysis. We report here the generation of a conditional floxed allele of Plexin-B1 (gene symbol Plxnb1) in mouse using CRISPR/Cas9 technology to insert two loxP sites flanking critical exons. Targeting reagents (Cas9 protein, sgRNAs, ssODNs) were delivered into single-cell embryos by electroporation. After screening a total of 128 mouse pups by PCR and Sanger sequencing, two mice were identified carrying both loxP sites in the targeted Plxnb1 locus (success rate ~ 1.6%). The usage of Alt-R modified ssODNs increased targeting frequencies at one loxP site, but not the other. We also tested homology directed repair (HDR) enhancer V2 reagent, but addition of the enhancer reduced the viability of mouse embryos. The Plxnb1^flox allele was successfully transmitted through the germline in Mendelian ratios, and effective excision of the floxed region was confirmed by breeding with Cre recombinase strains.

To identify novel Six1-interacting proteins, we previously screened the fly interactome for Sine oculis-binding partners whose orthologues are also expressed in Xenopus embryos. We identified a zinc-finger MYM-containing protein—Zmym2—based on its sequence similarity in a few domains also found in the Drosophila and vertebrate Sine oculis-binding proteins (Sobp). Because recent studies established Zmym2 as a transcriptional repressor that interacts with Six4 during renal development, herein we assessed whether it interacts with Six1, can modify Six1's transcriptional activity, and is involved in cranial neural crest or placode gene expression. Although during early development Zmym2 is expressed in many of the same tissues as Six1 and contains several domains also found in Sobp, we did not detect any interaction by co-immunoprecipitation and did not detect any effect on Six1 + Eya1 transcriptional activity in cultured cells. Nonetheless, increasing the level of Zmym2 in embryos resulted in broader expression domains of neural border, neural tube and neural crest genes, and smaller placode gene domains. These results suggest that although Zmym2 is unlikely to be a bone fide Six1 interacting protein, it appears to indirectly antagonize Six1 function during cranial placode development, promoting neural plate and neural crest gene expression.

Single-cell RNA sequencing (scRNA-seq) is a rapidly developing and useful technique for elucidating biological mechanisms and characterizing individual cells. Tens of millions of patients worldwide suffer from heart injuries and other types of heart disease. Neonatal mammalian hearts and certain adult vertebrate species, such as zebrafish, can fully regenerate after myocardial injury. However, the adult mammalian heart is unable to regenerate the damaged myocardium. scRNA-seq provides many new insights into pathological and normal hearts and facilitates our understanding of cellular responses to cardiac injury and repair at different stages, which may provide critical clues for effective therapies for adult heart regeneration. In this review, we summarize the application of scRNA-seq in heart development and regeneration and describe how important molecular mechanisms can be harnessed to promote heart regeneration.

Circadian clocks have a fundamental role in many physiological processes. Bmal1 (basic helix–loop–helix ARNT like 1) is a central master circadian clock gene. The global Bmal1 knockout mice were shown to have a loss of circadian rhythms, acceleration of aging, and shortened life span. However, global Bmal1 knockout mice did not exactly reflect the Bmal1 function in specific cell or tissue types. To address the importance of circadian rhythms in macrophages, we generated myeloid-specific Bmal1 knockout mice. The efficacy of Bmal1 gene deletion in macrophages was identified at DNA, transcription, protein levels, and function. In contrast to global Bmal1 knockout mice, Bmal1^flox/flox and Bmal1^mye−/− mice did not exhibit aging phenotypes. However, the deletion of Bmal1 resulted in a loss of rhythmic expression of the circadian genes in macrophages. RNA-Seq revealed that Bmal1 regulated the expression of cell death-related genes in macrophages. Furthermore, these genes have been identified as clock-controlled genes in rhythmic cell models, and Bmal1 controlled the rhythmic expression of these genes in macrophages. Finally, Bmal1 inhibited RSL3-induced ferroptosis in macrophages through Phgdh. In summary, the model of myeloid-specific Bmal1 knockout mice was successfully constructed, providing a tool for the study of the roles of Bmal1 in macrophages and the peripheral circadian clock. Meanwhile, Bmal1 regulates ferroptosis in macrophages.

Heterotaxy (HTX) is characterized by an abnormality in the organ arrangement along the Left-Right (LR) axis and is caused by the disruption of LR patterning in early development. LR asymmetry is critical for multiple organs. Specifically, proper LR patterning is crucial for cardiac function and is a cause of congenital heart disease (CHD). CACNA1G is a candidate gene identified in patients with CHD and HTX. This gene encodes a T-type, low-voltage-activated calcium channel and is a member of the Cav3.1 channel family. However, its function in cardiac or embryonic development remains unknown. Here, we show that abnormal cacna1g expression in Xenopus tropicalis recapitulates the HTX phenotype found in the patient cohort. By examining early LR patterning markers, including pitx2c and dand5, we discovered that both markers are expressed abnormally, suggesting that LR patterning is disrupted at the earliest stages of the LR signaling cascade. Since cilia have been described as key regulators of LR asymmetry, we checked the process of cilia formation in cacna1g crispants. The LR Organizer (LRO) contained reduced cilia quantity in the cacna1g crispants, which may explain the LR defects. In conclusion, the abnormal expression of cacna1g affects cilia in the LRO, leading to abnormal LR patterning and cardiac looping.