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Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal 1 – 3 . Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes—which are also cyclostomes—have only a single canal, which used to be regarded as a more primitive trait 1 , 4 . However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes 5 – 7 , which has left the evolutionary trajectory of the vertebrate inner ear unclear 8 . Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri . This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions—which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily—not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes. The differentiation of the inner ear in the lamprey Lethenteron camtschaticum and hagfish Eptatretus burgeri sheds light on the evolution of the semicircular canals of jawed vertebrates.

Background The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century. Results Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4 , a key regulatory gene, in the pharyngeal epithelium during early developmental stages. Conclusions Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys.

Background Vertebrates are characterized by possession of hypobranchial muscles (HBMs). Cyclostomes, or modern jawless vertebrates, possess a rudimentary and superficial HBM lateral to the pharynx, whereas the HBM in jawed vertebrates is internalized and anteroposteriorly specified. Precursor cells of the HBM, marked by expression of Lbx1 , originate from somites and undergo extensive migration before becoming innervated by the hypoglossal nerve. How the complex form of HBM arose in evolution is relevant to the establishment of the vertebrate body plan, but despite having long been assumed to be similar to that of limb muscles, modification of developmental mechanisms of HBM remains enigmatic. Results Here we characterize the expression of Lbx genes in lamprey and hagfish (cyclostomes) and catshark (gnathostome; jawed vertebrates). We show that the expression patterns of the single cyclostome Lbx homologue, Lbx-A , do not resemble the somitic expression of mammalian Lbx1 . Disruption of Lbx-A revealed that LjLbx-A is required for the formation of both HBM and body wall muscles, likely due to the insufficient extension of precursor cells rather than to hindered muscle differentiation. Both homologues of Lbx in the catshark were expressed in the somitic muscle primordia, unlike in amniotes. During catshark embryogenesis, Lbx2 is expressed in the caudal HBM as well as in the abdominal rectus muscle, similar to lamprey Lbx-A , whereas Lbx1 marks the rostral HBM and pectoral fin muscle. Conclusions We conclude that the vertebrate HBM primarily emerged as a specialized somatic muscle to cover the pharynx, and the anterior internalized HBM of the gnathostomes is likely a novelty added rostral to the cyclostome-like HBM, for which duplication and functionalization of Lbx genes would have been a prerequisite.

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)—a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species—a hagfish, a lamprey and a shark—encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates. Hox genes play a fundamental role in vertebrate development. Here, the authors show that, like in jawed vertebrates, in cyclostomes the onset of Hox gene expression is determined by their position in the Hox gene cluster.

Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx -deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin + fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.

Background The extant vertebrates include cyclostomes (lamprey and hagfish) and crown gnathostomes (jawed vertebrates), but there are various anatomical disparities between these two groups. Conspicuous in the gnathostomes is the neck, which occupies the interfacial domain between the head and trunk, including the occipital part of the cranium, the shoulder girdle, and the cucullaris and hypobranchial muscles (HBMs). Of these, HBMs originate from occipital somites to form the ventral pharyngeal and neck musculature in gnathostomes. Cyclostomes also have HBMs on the ventral pharynx, but lack the other neck elements, including the occipital region, the pectoral girdle, and cucullaris muscles. These anatomical differences raise questions about the evolution of the neck in vertebrates. Results In this study, we observed developing HBMs as a basis for comparison between the two groups and show that the arrangement of the head–trunk interface in gnathostomes is distinct from that of lampreys. Our comparative analyses reveal that, although HBM precursors initially pass through the lateral side of the pericardium in both groups, the relative positions of the pericardium withrespect to the pharyngeal arches differ between the two, resulting in diverse trajectories of HBMs in gnathostomes and lampreys. Conclusions We suggest that a heterotopic rearrangement of early embryonic components, including the pericardium and pharyngeal arches, may have played a fundamental role in establishing the gnathostome HBMs, which would also have served as the basis for neck formation in the jawed vertebrate lineage.

Background The skeletal musculature of gnathostomes, which is derived from embryonic somites, consists of epaxial and hypaxial portions. Some hypaxial muscles, such as tongue and limb muscles, undergo de-epithelialization and migration during development. Delamination and migration of these myoblasts, or migratory muscle precursors (MMPs), is generally thought to be regulated by hepatocyte growth factor (HGF) and receptor tyrosine kinase (MET) signaling. However, the prevalence of this mechanism and the expression patterns of the genes involved in MMP development across different vertebrate species remain elusive. Results We performed a comparative analysis of Hgf and Met gene expression in several vertebrates, including mouse, chicken, dogfish ( Scyliorhinus torazame) , and lamprey ( Lethenteron camtschaticum) . While both Hgf and Met were expressed during development in the mouse tongue muscle, and in limb muscle formation in the mouse and chicken, we found no clear evidence for the involvement of HGF/MET signaling in MMP development in shark or lamprey embryos. Conclusions Our results indicate that the expressions and functions of both Hgf and Met genes do not represent shared features of vertebrate MMPs, suggesting a stepwise participation of HGF/MET signaling in MMP development during vertebrate evolution.

Fibrocartilaginous entheses consist of four graded tissue layers including tendon, the unmineralized and mineralized fibrocartilage, and subchondral bone with varying degrees of stiffness. Here we examined the functional role of sclerostin that is expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of the unmineralized fibrocartilage and parallel replacement of epiphyseal hyaline cartilage by bone, the unmineralized fibrocartilage re-expanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin in the bottom of the mineralized fibrocartilage adjacent to subchondral bone. In Scx deficient mice with less mechanical loading due to defects of the Achilles tendon, the number of sclerostin+ fibrochondrocytes was significantly reduced in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, coding for sclerostin, caused increased mineral density in the mineralized zones of the fibrocartilaginous enthesis. Atomic force microscopy analysis revealed the higher stiffness of fibrocartilage. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of the fibrocartilaginous enthesis.Competing Interest StatementThe authors have declared no competing interest.

Runt-related transcription factor 2 (RUNX2) is essential for skeletogenesis, and mutations in its gene cause cleidocranial dysplasia (CCD), an autosomal dominant skeletal disorder. The evolutionarily conserved 128-amino acid Runt homology domain (RHD) of human RUNX2 is essential for DNA binding and heterodimerization, and serves as a mutation hotspot associated with severe CCD phenotypes. To elucidate the functional impact of pathogenic RHD mutations in vivo, we generated two novel mouse lines: one carrying a missense mutation, c.695G>A (p.R232Q) (Runx2m/+), corresponding to the human RUNX2 c.674G>A (p.R225Q), and the other harboring a frameshift mutation, c.697_698delGA (p.E233TfsTer9) (Runx2112/+), causing a premature stop codon. Homozygous Runx2m/mand Runx2112/112 mice lacked membranous ossification, whereas heterozygous Runx2m/+ and Runx2112/+ mice displayed typical CCD-like skeletal features, including an open anterior fontanelle and clavicle hypoplasia. Unexpectedly, heterozygotes carrying pathogenic mutations in RHD developed an accessory root-like protrusion at the furcation of three-rooted maxillary first molars, representing a previously unrecognized dental phenotype during root development. Dual luciferase assays revealed impaired transactivation of the p.R232Q mutant Runx2 on the osteocalcin enhancer/promoter. Wild-type Runx2 was robustly expressed in osteoblasts and hypertrophic chondrocytes during bone formation, but the mutant Runx2 exhibited reduced expression in hypertrophic chondrocytes and partially impaired nuclear localization, resulting in arrested osteoblast and chondrocyte maturation. Our mutant mouse model provides a valuable in vivo platform to study CCD pathogenesis, mechanisms of tooth root furcation, and therapeutic interventions targeting dysfunctional RHD.

Background Most evidence regarding lateral lymph node dissection for rectal cancer is from expert settings. This study aimed to evaluate the safety and efficacy of this procedure in a practice-based cohort. Methods A total of 383 patients who were diagnosed with stage II–III mid-to-low rectal cancer between 2010 and 2019 and underwent primary resection with curative intent at a general surgery unit were retrospectively reviewed. After propensity matching, 144 patients were divided into the following groups for short- and long-term outcome evaluation: mesorectal excision with lateral lymph node dissection (n = 72) and mesorectal excision (n = 72). Results This practice-based cohort was characterized by a high pT4 (41.6%) and R1 resection (10.4%) rate. Although the operative time was longer in the lateral dissection group (349 min vs. 237 min, p  < 0.001), postoperative complications (19.4% vs. 16.7%, p  = 0.829), and hospital stay (18 days vs. 22 days, p  = 0.059) did not significantly differ; 5-year relapse-free survival (62.5% vs. 66.4%, p  = 0.378), and cumulative local recurrence (9.7% vs. 15.3%, p = 0.451) were also in the same range in both groups. In the seven locally recurrent cases in the lateral dissection group, four had undergone R1 resection. Conclusions Lateral lymph node dissection was found to be safe in this practice-based cohort; however, the local control effect was not obvious. To maximize the potential merits of lateral lymph node dissection, strategies need to be urgently established to avoid R1 resection in clinical practice.