Masakatsu Watanabe

Nonchordate gap junction channels are formed by innexins, yet the structures and subunit assembly mechanisms of insect innexins remain unknown. We determined cryoelectron microscopy structures of AGAP001487, an innexin from the malaria mosquito Anopheles gambiae , reconstituted in lipid nanodiscs. The wild-type protein forms a rotationally symmetric heptameric hemichannel with an ordered N-terminal ring stabilized by intersubunit hydrogen bonds. Whereas deletion of a single N-terminal residue preserves channel activity, removal of two or more residues markedly impairs function. Consistent with these functional defects, the structure lacking two N-terminal residues exhibits disrupted rotational symmetry, extensive N-terminal disorder, pore collapse, and altered pore–lipid interactions. In addition, two-dimensional class averages revealed a minor population of hexameric particles, consistent with a potential assembly intermediate. Together, these structures support a stepwise assembly pathway in which intersubunit hydrogen bonding stabilizes N-terminal ring formation as a maturation step leading to a fully symmetric heptameric channel. Our findings identify N-terminal integrity as a critical determinant of oligomer maturation and channel function and provide mechanistic insight into how defined oligomeric assemblies are established in large-pore channel families.

The diverse pigmentation patterns of animals are crucial for predation avoidance and behavioral display, yet mechanisms underlying this diversity remain poorly understood. In zebrafish, Turing models have been proposed to explain stripe patterns, but it is unclear if they apply to other fishes. In anemonefish ( Amphiprion ocellaris) , we identified gja5b , a gene orthologous to zebrafish leopard and encoding a connexin involved in pigment cell communication, as responsible for the Snowflake phenotype. Using CRISPR/Cas9 and transgenesis, we recapitulate the Snowflake phenotype and show expression of gja5b in iridophores. A matching allele was recovered in zebrafish, revealing complementary requirements in both species. Our findings highlight conserved roles of gap junction mediated communication in pigment patterning across divergent teleosts.

Haematopoietic stem and progenitor cells (HSPCs) derive from a subset of endothelial cells (ECs), known as haemogenic ECs by the process of endothelial-to-haematopoietic transition (EHT). Although many factors involved in EHT have been elucidated, we still have a poor understanding of the temporal regulation of this process. Mitochondrial-derived reactive oxygen species (ROS) have been shown to stabilise the hypoxia-inducible factor 1/2α (Hif1/2α) proteins, allowing them to positively regulate EHT. Here, we show a developmental delay in EHT and HSPC induction in a connexin (cx)41.8 (orthologous to mammalian CX40) gap junction mutant, in zebrafish. In mammalian cells, CX40 has been shown to localise to the mitochondria. We demonstrate that Cx41.8 is important for the correct temporal generation of mitochondrial ROS, which stabilise the Hif pathway, allowing for the subsequent specification of the haemogenic endothelium. Taken together, our data indicate that Cx41.8 governs the correct temporal induction of HSPCs.

The mitochondrial translation system contains two ribosome rescue factors, ICT1 and MTRFR (C12orf65), which hydrolyze peptidyl‐tRNA in stalled ribosomes. ICT1 also functions as a ribosomal protein of the mitochondrial large ribosomal subunit (mtLSU) in mice and humans, and its deletion is lethal. In contrast, MTRFR does not share this role. Although loss‐of‐function mutations in MTRFR have been linked to human mitochondrial diseases, data on this association in other vertebrates are lacking. Here, attempts to generate Mtrfr knockout mice were unsuccessful. However, knockout zebrafish lines were successfully generated for both ict1 and mtrfr (ict1^−/− and mtrfr^−/−). Both knockout lines appeared healthy and fertile. ict1^−/−, mtrfr^−/−, and wild‐type adult caudal fin cells showed significant differences in mitochondrial morphology. The ict1 deletion affected the network properties more than the number of individuals and networks, whereas the mtrfr deletion exhibited the opposite effect. Additionally, the survival rates of the knockout line larvae were significantly lower than those of the wild‐type larvae under starvation conditions. These results suggest that ict1 and mtrfr are required for survival under specific stress conditions, whereas ict1^−/− and mtrfr^−/− involve different compensatory mechanisms in response to loss of either factor under nonstress conditions. Ict1 proteins from all teleosts, including zebrafish, lack the N‐terminal mtLSU‐binding motif found in most metazoans, suggesting that Ict1 does not function as a ribosomal protein in teleosts. Thus, Mtrfr may partially compensate for the loss of Ict1. In conclusion, zebrafish appear to exemplify a limited category of vertebrates capable of enduring genetic abnormalities in ict1 or mtrfr.

While depolarization of the neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here, we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their postsynaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

Scoliosis is a condition where the spine curves sideways, unique to humans due to their upright posture. However, the cause of this disease is not well understood because it is challenging to find a model for experimentation. This study aimed to create a model for human idiopathic scoliosis by manipulating the function of mechanosensitive channels called Piezo channels in zebrafish. Zebrafish were chosen because they experience similar biomechanical forces to humans, particularly in relation to the role of mechanical force in scoliosis progression. Here we describe piezo1 and piezo2a are involved in bone formation, with a double knockout resulting in congenital systemic malformations. However, an in-frame mutation of piezo1 led to fully penetrant juvenile-onset scoliosis, bone asymmetry, reduced tissue mineral density, and abnormal intervertebral discs—resembling non-congenital scoliosis symptoms in humans. These findings suggest that functional Piezo channels responding to mechanical forces are crucial for bone formation and maintaining spine integrity, providing insights into skeletal disorders.

Scoliosis is a condition where the spine curves sideways, unique to humans due to their upright posture. However, the cause of this disease is not well understood because it’s challenging to find a model for experimentation. This study aimed to create a model for human idiopathic scoliosis by manipulating the function of mechanosensitive channels called Piezo channels in zebrafish. Zebrafish were chosen because they experience similar biomechanical forces to humans, particularly in relation to the role of mechanical force in scoliosis progression. Here we describe piezo1 and piezo2a are involved in bone formation, with a double knockout resulting in congenital systemic malformations. However, an in-frame mutation of piezo1 led to fully penetrant juvenile-onset scoliosis, bone asymmetry, reduced tissue mineral density, and abnormal intervertebral discs-resembling non-congenital scoliosis symptoms in humans. These findings suggest that functional Piezo channels responding to mechanical forces are crucial for bone formation and maintaining spine integrity, providing insights into skeletal disorders.

While depolarization of neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their post-synaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

Haematopoietic stem and progenitor cells (HSPCs) derive from a subset of endothelial cells (ECs), known as haemogenic ECs by the process of endothelial-to-haematopoietic transition (EHT). Although many factors involved in EHT have been elucidated, we still have a poor understanding of the temporal regulation of this process. Mitochondrial-derived reactive oxygen species (ROS) have been shown to stabilise hypoxia-inducible factors 1/2α (Hif1/2α), allowing them to positively regulate EHT. Here, we show a developmental delay in EHT and HSPC induction in a gap junction mutant, connexin (cx)41 . 8 (orthologous to mammalian CX40 ), in zebrafish. In mammalian cells, CX40 has been shown to localise to the mitochondria. We demonstrate that Cx41.8 is important for the correct temporal generation of mitochondrial ROS, which stabilise the Hif pathway, allowing for the subsequent specification of the haemogenic endothelium. Taken together, our data indicate that Cx41.8 mediates the correct induction of HSPCs.