Masaki Takechi’s research while affiliated with Juntendo University and other places

The anatomical innovation of sound-producing organs, which gives rise to a wide variety of sound signals, is one of the most fundamental factors leading to the explosive speciation of modern birds. Despite being a key clue to resolving the homology of sound-controlling muscles among birds, only few studies have explored the embryonic development of syringeal muscles. Using serial histological sections and immunohistochemistry, we described the three-dimensional anatomy and development of the cartilage, muscle, and innervation pattern of the tracheobronchi in three avian species: domestic fowls, cockatiels, and zebra finches. Crucially, the muscle primordia of the syringeal and tracheobronchial muscles develop from the caudal end of the lateral tracheal muscle in cockatiels and zebra finches. Furthermore, the tracheobronchial and syringeal muscle primordia of the zebra finches are subsequently split during embryonic development. Based on our findings on the identity of muscle primordia development and innervation pattern of the hypoglossal nerve between the cockatiels and zebra finches, we suggest that the muscle component traditionally documented as the superficial syringeal muscle in parrots is homologous to the tracheobronchial and ventral syringeal muscles of the zebra finches. These facts not only orchestrate the terminological discrepancies among previous studies, but also suggest that the syringeal muscles were acquired by the anatomical innovation of the lateral tracheal muscles in the common ancestor of the parrots and passerines, and further compartmentalized in the passerines, perhaps leading to a wide song repertoire for acoustic communication.

Sonic hedgehog ( Shh ) is expressed in the oropharyngeal epithelium, including the frontonasal ectodermal zone (FEZ), which is defined as the boundary between Shh and Fgf8 expression domains in the frontonasal epithelium. To investigate the role of SHH signaling from the oropharyngeal epithelium, we generated mice in which Shh expression is specifically deleted in the oropharyngeal epithelium ( Isl1‐Cre; Shh f/f ). In the mutant mouse, Shh expression was excised in the oropharyngeal epithelium as well as FEZ and ventral forebrain, consistent with the expression pattern of Isl1 . Isl1‐Cre; Shh f/f mice exhibited a complete loss of lower jaw components and a malformed upper jaw with defects in the cranial base and secondary palate. Massive cell death was observed in the mandibular process at embryonic day (E) 9.5 and E10.5, while mild cell death was observed in the lambdoidal region (the fusion area in the maxillary, lateral nasal, and medial nasal processes) at E10.5. An RNA‐seq analysis revealed that Satb2 , a gene involved in cell survival during jaw formation, was downregulated in the lambdoidal region in Isl1‐Cre; Shh f/f mice. These results suggest that Shh expression in the FEZ is required for cell survival and skeletogenesis in the lambdoidal region during the development of the upper jaw and that the developmental control governed by SHH signaling is different between upper and lower jaws.

Background Mutations in genes encoding spliceosome components result in craniofacial structural defects in humans, referred to as spliceosomopathies. The SF3b complex is a crucial unit of the spliceosome, but model organisms generated through genetic modification of the complex do not perfectly mimic the phenotype of spliceosomopathies. Since the phenotypes are suggested to be determined by the extent of spliceosome dysfunction, an alternative experimental system that can seamlessly control SF3b function is needed. Methods To establish another experimental system for model organisms elucidating relationship between spliceosome function and human diseases, we administered Pladienolide‐B (PB), a SF3b complex inhibitor, to mouse and zebrafish embryos and assessed resulting phenotypes. Results PB‐treated mouse embryos exhibited neural tube defect and exencephaly, accompanied by apoptosis and reduced cell proliferation in the neural tube, but normal structure in the midface and jaw. PB administration to heterozygous knockout mice of Sf3b4 , a gene coding for a SF3b component, influenced the formation of cranial neural crest cells (CNCCs). Despite challenges in continuous PB administration and a high death rate in mice, PB was stably administered to zebrafish embryos, resulting in prolonged survival. Brain, cranial nerve, retina, midface, and jaw development were affected, mimicking spliceosomopathy phenotypes. Additionally, alterations in cell proliferation, cell death, and migration of CNCCs were detected. Conclusions We demonstrated that zebrafish treated with PB exhibited phenotypes similar to those observed in human spliceosomopathies. This experimental system may serve as a valuable research tool for understanding spliceosome function and human diseases.

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of the glomeruli has mainly been evaluated using transmission electron microscopy. However, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we propose an AT workflow optimized for observing complete serial sections of the whole glomerulus, shared several analytical examples using the optimized AT workflow, and demonstrate the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells.

Snakes show remarkably deviated “body plan” from other squamate reptiles. In addition to limb loss, they have accomplished enormous anatomical specialization of the skull associated with the pit organs and the reduction of the tympanic membranes and auditory canals in the outer ears. Despite being the most diverse group of snakes, our knowledge of the embryonic staging for organogenesis and cranial ossification has been minimal for Colubridae. Therefore, in the present observation, we provide the first embryonic description of the Japanese rat snake Elaphe climacophora. We based our study on the Standard Event System (SES) for external anatomical characters and on a description of the cranial ossification during post‐ovipositional development. We further estimated the relative ossification timing of each cranial bony element and compared it with that of selected other snakes, lizards, turtles, and crocodilians. The present study shows that the relative ossification timing of the palatine and pterygoid bones is relatively early in squamates when compared to other reptiles, implying the developmental integration as the palate–pterygoid complex in this clade and functional demands for the unique feeding adaptation to swallow large prey with the help of their large palatine and pterygoid teeth. Furthermore, unlike in species with pit organs, the prootic bone of Ela. climacophora is expanded to provide articulation with the supratemporal, thereby contributing to the hearing system by detecting substrate vibration. We also demonstrate that the relative timing of the prootic ossification is significantly accelerated in colubrids compared to snakes with pit organs. Our finding suggests that the temporal changes of the prootic ossification underpin the evolution of the perception of the ground‐bourne sound signals among snakes.

Background The hyolaryngeal apparatus generates biosonar pulses in the laryngeally echolocating bats. The cartilage and muscles comprising the hyolarynx of laryngeally echolocating bats are morphologically modified compared to those of non-bat mammals, as represented by the hypertrophied intrinsic laryngeal muscle. Despite its crucial contribution to laryngeal echolocation, how the development of the hyolarynx in bats differs from that of other mammals is poorly documented. The genus Rhinolophus is one of the most sophisticated laryngeal echolocators, with the highest pulse frequency in bats. The present study provides the first detailed description of the three-dimensional anatomy and development of the skeleton, cartilage, muscle, and innervation patterns of the hyolaryngeal apparatus in two species of rhinolophid bats using micro-computed tomography images and serial tissue sections and compares them with those of laboratory mice. Furthermore, we measured the peak frequency of the echolocation pulse in active juvenile and adult individuals to correspond to echolocation pulses with hyolaryngeal morphology at each postnatal stage. Results We found that the sagittal crests of the cricoid cartilage separated the dorsal cricoarytenoid muscle in horseshoe bats, indicating that this unique morphology may be required to reinforce the repeated closure movement of the glottis during biosonar pulse emission. We also found that the cricothyroid muscle is ventrally hypertrophied throughout ontogeny, and that the cranial laryngeal nerve has a novel branch supplying the hypertrophied region of this muscle. Our bioacoustic analyses revealed that the peak frequency shows negative allometry against skull growth, and that the volumetric growth of all laryngeal cartilages is correlated with the pulse peak frequency. Conclusions The unique patterns of muscle and innervation revealed in this study appear to have been obtained concomitantly with the acquisition of tracheal chambers in rhinolophids and hipposiderids, improving sound intensity during laryngeal echolocation. In addition, significant protrusion of the sagittal crest of the cricoid cartilage and the separated dorsal cricoarytenoid muscle may contribute to the sophisticated biosonar in this laryngeally echolocating lineage. Furthermore, our bioacoustic data suggested that the mineralization of these cartilages underpins the ontogeny of echolocation pulse generation. The results of the present study provide crucial insights into how the anatomy and development of the hyolaryngeal apparatus shape the acoustic diversity in bats.

Background The hyolaryngeal apparatus generates biosonar pulses in the laryngeally echolocating bats. The cartilage and muscles comprising the hyolarynx of laryngeally echolocating bats are morphologically modified compared to those of non-bat mammals, as represented by the hypertrophied intrinsic laryngeal muscle. Despite its crucial contribution to laryngeal echolocation, how the development of the hyolarynx in bats differs from that of other mammals is poorly documented. The genus Rhinolophus is one of the most sophisticated laryngeal echolocators, with the highest pulse frequency in bats. The present study provides the first detailed description of the three-dimensional anatomy and development of the skeleton, cartilage, muscle, and innervation patterns of the hyolaryngeal apparatus in two species of rhinolophid bats using micro-computed tomography images and serial tissue sections and compares them with those of laboratory mice. Furthermore, we measured the peak frequency of the echolocation pulse in active juvenile and adult individuals to correspond to echolocation pulses with hyolaryngeal morphology at each postnatal stage. Results We found that the sagittal crests of the cricoid cartilage separated the dorsal cricoarytenoid muscle in horseshoe bats, indicating that this unique morphology may be required to reinforce the repeated closure movement of the glottis during biosonar pulse emission. We also found that the cricothyroid muscle is ventrally hypertrophied throughout ontogeny, and that the cranial laryngeal nerve has a novel branch supplying the hypertrophied region of this muscle. Our bioacoustic analyses revealed that the peak frequency shows negative allometry against skull growth, and that the volumetric growth of all laryngeal cartilages is correlated with the pulse peak frequency. Conclusions The unique patterns of muscle and innervation revealed in this study appear to have been obtained concomitantly with the acquisition of tracheal chambers in rhinolophids and hipposiderids, improving sound intensity during laryngeal echolocation. In addition, significant protrusion of the sagittal crest of the cricoid cartilage and the separated dorsal cricoarytenoid muscle may contribute to the sophisticated biosonar in this laryngeally echolocating lineage. Furthermore, our bioacoustic data suggested that the mineralization of these cartilages underpins the ontogeny of echolocation pulse generation. The results of the present study provide crucial insights into how the anatomy and development of the hyolaryngeal apparatus shape the acoustic diversity in bats.

Fibroblast growth factor (FGF) signaling plays essential roles in various biological events. FGF18 is one of the ligands to be associated with osteogenesis, chondrogenesis and bone healing. The mouse critical-sized calvarial defect healing induced by the bone morphogenetic protein 2 (BMP2)-hydrogel is stabilized when FGF18 is added. Here, we aimed to investigate the role of FGF18 in the calvarial bone healing model. We first found that FGF18 + BMP2 hydrogel application to the calvarial bone defect increased the expression of anti-inflammatory markers, including those related to tissue healing M2 macrophage (M2-Mø) prior to mineralized bone formation. The depletion of macrophages with clodronate liposome hindered the FGF18 effect. We then examined how FGF18 induces M2-Mø polarization by using mouse primary bone marrow (BM) cells composed of macrophage precursors and BM stromal cells (BMSCs). In vitro studies demonstrated that FGF18 indirectly induces M2-Mø polarization by affecting BMSCs. Whole transcriptome analysis and neutralizing antibody treatment of BMSC cultured with FGF18 revealed that chemoattractant chemokine (c-c motif) ligand 2 (CCL2) is the major mediator for M2-Mø polarization. Finally, FGF18-augmented activity toward favorable bone healing with BMP2 was diminished in the calvarial defect in Ccr2-deleted mice. Altogether, we suggest a novel role of FGF18 in M2-Mø modulation via stimulation of CCL2 production in calvarial bone healing.

Bats can be phylogenetically classified into three major groups: pteropodids, rhinolophoids, and yangochiropterans. While rhinolophoids and yangochiropterans are capable of laryngeal echolocation, pteropodids lack this ability. Delicate ear movements are essential for echolocation behavior in bats with laryngeal echolocation. Caudal auricular muscles, especially the cervicoauricularis group, play a critical role in such ear movements. Previously, caudal auricular muscles were studied in three species of bats with laryngeal echolocation, but to our knowledge, there have been no studies on non-laryngeal echolocators, the pteropodids. Here, we describe the gross anatomy of the cervicoauricularis muscles and their innervation in Cynopterus sphinx by using diffusible iodine-based contrast-enhanced computed tomography and 3D reconstructions of immunohistochemically stained serial sections. A previous study on bats with laryngeal echolocation reported that rhinolophoids have four cervicoauricularis muscles and yangochiropterans have three. We observed three cervicoauricularis muscles in the pteropodid C. sphinx. The number of cervicoauricularis muscles and their innervation pattern were comparable to those of non-bat boreoeutherian mammals and yangochiropterans, suggesting that pteropodids, and yangochiropterans maintain the general condition of boreoeutherian mammals and that rhinolophoids have a derived condition. The unique nomenclature had been previously applied to the cervicoauricularis muscles of bats with laryngeal echolocation, but given the commonality between non-bat laurasiatherians and bats, with the exception of rhinolophoids, maintaining the conventional nomenclature (i.e., M. cervicoauricularis superficialis, M. cervicoauricularis medius, and M. cervicoauricularis profundus) is proposed for bats.

Bats have undergone one of the most drastic limb innovations in vertebrate history, associated with the evolution of powered flight. Knowledge of the genetic basis of limb organogenesis in bats has increased but little has been documented regarding the differences between limb organogenesis in bats and that of other vertebrates. We conducted embryological comparisons of the timelines of limb organogenesis in 24 bat species and 72 non-bat amniotes. In bats, the time invested for forelimb organogenesis has been considerably extended and the appearance timing of the forelimb ridge has been significantly accelerated, whereas the timing of the finger and first appearance of the claw development has been delayed, facilitating the enlargement of the manus. Furthermore, we discovered that bats initiate the development of their hind limbs earlier than their forelimbs compared to other placentals. Bat neonates are known to be able to cling continuously with their well-developed foot to the maternal bodies or habitat substrates soon after birth. We suggest that this unique life history of neonates, which possibly coevolved with powered flight, has driven the accelerated development of the hind limb and precocious foot.