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Pericentric heterochromatin is a critical component of chromosomes marked by histone H3 K9 (H3K9) methylation 1-3 . However, what recruits H3K9-specific histone methyltransferases to pericentric regions in vertebrates remains unclear 4 , as does why pericentric regions in different species share the same H3K9 methylation mark despite lacking highly conserved DNA sequences 2,5 . Here we show that zinc-finger proteins ZNF512 and ZNF512B specifically localize at pericentric regions through direct DNA binding. Notably, both ZNF512 and ZNF512B are sufficient to initiate de novo heterochromatin formation at ectopically targeted repetitive regions and pericentric regions, as they directly recruit SUV39H1 and SUV39H2 (SUV39H) to catalyse H3K9 methylation. SUV39H2 makes a greater contribution to H3K9 trimethylation, whereas SUV39H1 seems to contribute more to silencing, probably owing to its preferential association with HP1 proteins. ZNF512 and ZNF512B from different species can specifically target pericentric regions of other vertebrates, because the atypical long linker residues between the zinc-fingers of ZNF512 and ZNF512B offer flexibility in recognition of non-consecutively organized three-nucleotide triplets targeted by each zinc-finger. This study addresses two long-standing questions: how constitutive heterochromatin is initiated and how seemingly variable pericentric sequences are targeted by the same set of conserved machinery in vertebrates., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

For postmetamorphic specimens of amphioxus (Cephalochordata), serial block-face scanning electron microscopy (SBSEM) is used to describe the long-ignored Rohde-like cells (RLCs) at the extreme posterior end of the dorsal nerve cord. These cells, numbering about three dozen in all, are divisible into a group with larger diameters running near the dorsal side of the cord and a more ventral group with smaller diameters closely associated with the central canal of the neurocoel. It is possible that the smaller ventral cells might be generated at the ependymal zone of the dorsal nerve cord and later migrate to a dorsal position, although a functional reason for this remains a mystery. All the RLCs have conspicuous regions of microvilli covering as much as 40% of their surface; limited data (by others) on the more anterior bona fide Rohde cells also indicate an extensive microvillar surface. Thus, both the RLCs and the better-known Rohde cells appear to be rhabdomeric photoreceptors, although a specific function for this feature is currently unknown. Even more perplexingly, although the Rohde cells are quintessential neurons extending giant processes, each RLC comprises a perikaryon that does not bear any neurites., (© 2024 The Author(s). The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cystatins comprise a vast superfamily of evolutionary conserved proteins, predominantly recognized for their roles as endogenous inhibitors by regulating the activity of cysteine proteases. Emerging lines of research evidence also provides insight into their alternative roles in a spectrum of biological and pathological processes, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. Nowadays, various type-1 cystatins (stefins) have been demonstrated among a variety of discovered vertebrate groups, while little is known about the related homologue in cephalochordate amphioxus, which are repositioned at the base of the chordate phylum. In the present study, a single type-1 cystatin homologue in Branchiostoma japonicum was first successfully cloned and designated as Bjcystatin-1. The deduced Bjcystatin-1 protein is structurally characterized by the presence of typical wedge-shaped cystatin features, including the 'QxVxG' and 'Px' motif, as well as the conserved N-terminal glycine residue. Phylogenomic analyses utilizing different cystatin counterparts affirmed the close evolutionary relationship of Bjcystatin-1 and type-1 cystatin homologue. Bjcystatin-1 was predominantly expressed in the gills and hind-gut in a tissue-specific pattern, and its expression was remarkably up-regulated in response to challenge with bacteria or their signature molecules LPS and LTA, suggesting the involvement in immune response. Additionally, the recombinant Bjcystatin-1 (rBjcystatin-1) protein showed significant inhibitory activity towards papain and binding ability to LPS and LTA, indicating its hypothesized role as a pattern recognition receptor in immune response. Subcellular localization results also showed that Bjcystatin-1 was located in the cytoplasm and nucleus, and its overexpression could attenuate the activation of LPS-induced nuclear transcription factors NF-κB. Taken together, our study suggests that amphioxus Bjcystatin-1 acts as a dual role in protease inhibitor and an immunocompetent factor, providing new insights into the immune defense effect of type-1 cystatin in amphioxus., (Copyright © 2024. Published by Elsevier Ltd.)