Pathogenicity and transmissibility of bovine H5N1 influenza virus.

Highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses occasionally infect, but typically do not transmit, in mammals. In the Spring of 2024, an unprecedented outbreak of HPAI H5N1 in bovine herds occurred in the US, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk1-4. Here, we characterized an HPAI H5N1 virus isolated from infected cow milk in mice and ferrets. Like other HPAI H5N1 viruses, the bovine H5N1 virus spread systemically, including to the mammary glands of both species; however, this tropism was also observed for an older HPAI H5N1 virus isolate. Importantly, bovine HPAI H5N1 virus bound to sialic acids expressed in human upper airways and inefficiently transmitted to exposed ferrets (one of four exposed ferrets seroconverted without virus detection). Bovine HPAI H5N1 virus thus possesses features that may facilitate infection and transmission in mammals.

Characterization of a human H3N8 influenza virus.

BACKGROUND: In 2022 and 2023, novel reassortant H3N8 influenza viruses infected three people, marking the first human infections with viruses of this subtype. METHODS: Here, we generated one of these viruses (A/Henan/4-10CNIC/2022; hereafter called A/Henan/2022 virus) by using reverse genetics and characterized it. FINDINGS: In intranasally infected mice, reverse genetics-generated A/Henan/2022 virus caused weight loss in all five animals (one of which had to be euthanized) and replicated efficiently in the respiratory tract. Intranasal infection of ferrets resulted in minor weight loss and moderate fever but no mortality. Reverse genetics-generated A/Henan/2022 virus replicated efficiently in the upper respiratory tract of ferrets but was not detected in the lungs. Virus transmission via respiratory droplets occurred in one of four pairs of ferrets. Deep-sequencing of nasal swab samples from inoculated and exposed ferrets revealed sequence polymorphisms in the haemagglutinin protein that may affect receptor-binding specificity. We also tested 90 human sera for neutralizing antibodies against reverse genetics-generated A/Henan/2022 virus and found that some of them possessed neutralizing antibody titres, especially sera from older donors with likely exposure to earlier human H3N2 viruses. INTERPRETATION: Our data demonstrate that reverse genetics-generated A/Henan/2022 virus is a low pathogenic influenza virus (of avian influenza virus descent) with some antigenic resemblance to older human H3N2 viruses and limited respiratory droplet transmissibility in ferrets. FUNDING: This work was supported by the Japan Program for Infectious Diseases Research and Infrastructure (JP23wm0125002), and the Japan Initiative for World-leading Vaccine Research and Development Centers (JP233fa627001) from the Japan Agency for Medical Research and Development (AMED).

The mammalian midbody and midbody remnant are assembly sites for RNA and localized translation.

Long ignored as a vestigial remnant of cytokinesis, the mammalian midbody (MB) is released post-abscission inside large extracellular vesicles called MB remnants (MBRs). Recent evidence suggests that MBRs can modulate cell proliferation and cell fate decisions. Here, we demonstrate that the MB matrix is the site of ribonucleoprotein assembly and is enriched in mRNAs that encode proteins involved in cell fate, oncogenesis, and pluripotency, which we are calling the MB granule. Both MBs and post-abscission MBRs are sites of spatiotemporally regulated translation, which is initiated when nascent daughter cells re-enter G1 and continues after extracellular release. MKLP1 and ARC are necessary for the localization and translation of RNA in the MB dark zone, whereas ESCRT-III is necessary to maintain translation levels in the MB. Our work reveals a unique translation event that occurs during abscission and within a large extracellular vesicle.

Molecular development of chondrichthyan claspers and the evolution of copulatory organs.

The earliest known vertebrate copulatory organs are claspers, paired penis-like structures that are associated with evolution of internal fertilization and viviparity in Devonian placoderms. Today, only male chondrichthyans possess claspers, which extend from posterior pelvic fins and function as intromittent organs. Here we report that clasper development from pelvic fins of male skates is controlled by hormonal regulation of the Sonic hedgehog (Shh) pathway. We show that Shh signalling is necessary for male clasper development and is sufficient to induce clasper cartilages in females. Androgen receptor (AR) controls the male-specific pattern of Shh in pelvic fins by regulation of Hand2. We identify an androgen response element (ARE) in the Hand2 locus and present biochemical evidence that AR can directly bind the Hand2 ARE. Together, our results suggest that the genetic circuit for appendage development evolved an androgen regulatory input, which prolonged signalling activity and drove clasper skeletogenesis in male fins.

Hox genes regulate digit patterning by controlling the wavelength of a Turing-type mechanism.

The formation of repetitive structures (such as stripes) in nature is often consistent with a reaction-diffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the Gli3-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton patterns suggests that the pentadactyl state has been achieved through modification of an ancestral Turing-type mechanism.

A natural deletion of the HoxC cluster in elasmobranch fishes.

Hox proteins are a metazoan-specific family of transcription factors that are required for developmental patterning. The genomic arrangement of Hox genes into four paralogous clusters is a primitive feature of jawed vertebrates. By using high-throughput sequencing, we demonstrate the absence of all HoxC transcripts from embryos of the shark Scyliorhinus canicula and the skate Leucoraja erinacea and the absence of all HoxC genes and two HoxC-associated microRNAs from the genome of L. erinacea. These data suggest a loss of the entire HoxC cluster in elasmobranch fishes and represent evidence for the natural deletion of an entire Hox cluster in vertebrates.

Appendage expression driven by the Hoxd Global Control Region is an ancient gnathostome feature.

The evolutionary transition of the fins of fish into tetrapod limbs involved genetic changes to developmental systems that resulted in novel skeletal patterns and functions. Approaches to understanding this issue have entailed the search for antecedents of limb structure in fossils, genes, and embryos. Comparative genetic analyses have produced ambiguous results: although studies of posterior Hox genes from homology group 13 (Hoxa-13 and Hoxd-13) reveal similarities in gene expression between the distal segments of fins and limbs, this functional homology has not been supported by genomic comparisons of the activity of their cis-regulatory elements, namely the Hoxd Global Control Region. Here, we show that cis-regulatory elements driving Hoxd gene expression in distal limbs are present in fish. Using an interspecies transgenesis approach, we find functional conservation between gnathostome Hoxd enhancers, demonstrating that orthologous sequences from tetrapods, zebrafish and skate can drive reporter gene expression in mouse limbs and zebrafish fins. Our results support the notion that some of the novelties associated with tetrapod limbs arose by modification of deeply conserved cis- and trans-acting mechanisms of Hox regulation in gnathostomes.

Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration.

Zebrafish heart regeneration occurs through the activation of cardiomyocyte proliferation in areas of trauma. Here, we show that within 3 hr of ventricular injury, the entire endocardium undergoes morphological changes and induces expression of the retinoic acid (RA)-synthesizing enzyme raldh2. By one day posttrauma, raldh2 expression becomes localized to endocardial cells at the injury site, an area that is supplemented with raldh2-expressing epicardial cells as cardiogenesis begins. Induced transgenic inhibition of RA receptors or expression of an RA-degrading enzyme blocked regenerative cardiomyocyte proliferation. Injured hearts of the ancient fish Polypterus senegalus also induced and maintained robust endocardial and epicardial raldh2 expression coincident with cardiomyocyte proliferation, whereas poorly regenerative infarcted murine hearts did not. Our findings reveal that the endocardium is a dynamic, injury-responsive source of RA in zebrafish, and indicate key roles for endocardial and epicardial cells in targeting RA synthesis to damaged heart tissue and promoting cardiomyocyte proliferation.

Shared developmental mechanisms pattern the vertebrate gill arch and paired fin skeletons.

Here, we describe the molecular patterning of chondrichthyan branchial rays (gill rays) and reveal profound developmental similarities between gill rays and vertebrate appendages. Sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8) regulate the outgrowth and patterning of the chondrichthyan gill arch skeleton, in an interdependent manner similar to their roles in gnathostome paired appendages. Additionally, we demonstrate that paired appendages and branchial rays share other conserved developmental features, including Shh-mediated mirror-image duplications of the endoskeleton after exposure to retinoic acid, and Fgf8 expression by a pseudostratified distal epithelial ridge directing endoskeletal outgrowth. These data suggest that the skeletal patterning role of the retinoic acid/Shh/Fgf8 regulatory circuit has a deep evolutionary origin predating vertebrate paired appendages and may have functioned initially in patterning pharyngeal structures in a deuterostome ancestor of vertebrates.

Chondrogenesis and homology of the visceral skeleton in the little skate, Leucoraja erinacea (Chondrichthyes: Batoidea).

Chondrichthyan fishes possess visceral skeletons that differ considerably, morphologically, from those of their sister taxon, the osteichthyans. Here, we use histological techniques and whole-mount skeletal preparations to visualize and describe the sequence of visceral skeletal condensation and chondrogenesis in a chondrichthyan, the little skate (Leucoraja erinacea). We demonstrate that visceral skeletal condensation begins rostrally, with the mandibular arch, and progresses caudally with the hyoid arch and posterior branchial arches condensing soon after. We provide a detailed account of the condensation and chondrogenesis of all major components of the L. erinacea visceral skeleton and discuss these data in the context of what is known from classical descriptions of chondrichthyan visceral skeletal development. Significant differences exist between the hypobranchial and basibranchial skeleton of L. erinacea and other chondrichthyan species, and the possible evolutionary and developmental significance of this is considered. We discuss the homology of the chondrichthyan hyoid arch and, based on patterns of mesenchymal condensation, we propose a model of condensation splitting and diversification that may account for the morphological diversification of gnathostome branchial arch derivatives. Finally, we suggest that the unique presence of certain visceral skeletal elements in chondrichthyans make oviparous chondrichthyans an ideal system for addressing questions of endoskeletal axial patterning during development.

An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish.

Comparative analyses of Hox gene expression and regulation in teleost fish and tetrapods support the long-entrenched notion that the distal region of tetrapod limbs, containing the wrist, ankle and digits, is an evolutionary novelty. Data from fossils support the notion that the unique features of tetrapod limbs were assembled over evolutionary time in the paired fins of fish. The challenge in linking developmental and palaeontological approaches has been that developmental data for fins and limbs compare only highly derived teleosts and tetrapods; what is lacking are data from extant taxa that retain greater portions of the fin skeletal morphology considered primitive to all bony fish. Here, we report on the expression and function of genes implicated in the origin of the autopod in a basal actinopterygian, Polyodon spathula. Polyodon exhibits a late-phase, inverted collinear expression of 5' HoxD genes, a pattern of expression long considered a developmental hallmark of the autopod and shown in tetrapods to be controlled by a 'digit enhancer' region. These data show that aspects of the development of the autopod are primitive to tetrapods and that the origin of digits entailed the redeployment of ancient patterns of gene activity.

Sonic hedgehog function in chondrichthyan fins and the evolution of appendage patterning.

The genetic mechanisms regulating tetrapod limb development are well characterized, but how they were assembled during evolution and their function in basal vertebrates is poorly understood. Initial studies report that chondrichthyans, the most primitive extant vertebrates with paired appendages, differ from ray-finned fish and tetrapods in having Sonic hedgehog (Shh)-independent patterning of the appendage skeleton. Here we demonstrate that chondrichthyans share patterns of appendage Shh expression, Shh appendage-specific regulatory DNA, and Shh function with ray-finned fish and tetrapods. These studies demonstrate that some aspects of Shh function are deeply conserved in vertebrate phylogeny, but also highlight how the evolution of Shh regulation may underlie major morphological changes during appendage evolution.

The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb.

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5' genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh(-/-) mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.

Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity.

Most current models propose Sonic hedgehog (Shh) as the primary determinant of anteroposterior development of amniote limbs. Shh protein is said to be required to direct the formation of skeletal elements and to specify digit identity through dose-dependent activation of target gene expression. However, the identity of genes targeted by Shh, and the regulatory mechanisms controlling their expression, remain poorly understood. Gli3 (the gene implicated in human Greig cephalopolysyndactyly syndrome) is proposed to negatively regulate Shh by restricting its expression and influence to the posterior mesoderm. Here we report genetic analyses in mice showing that Shh and Gli3 are dispensable for formation of limb skeletal elements: Shh(-/-) Gli3(-/-) limbs are distally complete and polydactylous, but completely lack wild-type digit identities. We show that the effects of Shh signalling on skeletal patterning and ridge maintenance are necessarily mediated through Gli3. We propose that the function of Shh and Gli3 in limb skeletal patterning is limited to refining autopodial morphology, imposing pentadactyl constraint on the limb's polydactyl potential, and organizing digit identity specification, by regulating the relative balance of Gli3 transcriptional activator and repressor activities.