Timing of organogenesis underscores the evolution of neonatal life histories and powered flight in 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 hindlimbs earlier than their forelimbs compared with 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 hindlimb and precocious foot.

Synchondrosis fusion contributes to the progression of postnatal craniofacial dysmorphology in syndromic craniosynostosis.

Syndromic craniosynostosis (CS) patients exhibit early, bony fusion of calvarial sutures and cranial synchondroses, resulting in craniofacial dysmorphology. In this study, we chronologically evaluated skull morphology change after abnormal fusion of the sutures and synchondroses in mouse models of syndromic CS for further understanding of the disease. We found fusion of the inter-sphenoid synchondrosis (ISS) in Apert syndrome model mice (Fgfr2S252W/+ ) around 3 weeks old as seen in Crouzon syndrome model mice (Fgfr2cC342Y/+ ). We then examined ontogenic trajectories of CS mouse models after 3 weeks of age using geometric morphometrics analyses. Antero-ventral growth of the face was affected in Fgfr2S252W/+ and Fgfr2cC342Y/+ mice, while Saethre-Chotzen syndrome model mice (Twist1+/- ) did not show the ISS fusion and exhibited a similar growth pattern to that of control littermates. Further analysis revealed that the coronal suture synostosis in the CS mouse models induces only the brachycephalic phenotype as a shared morphological feature. Although previous studies suggest that the fusion of the facial sutures during neonatal period is associated with midface hypoplasia, the present study suggests that the progressive postnatal fusion of the cranial synchondrosis also contributes to craniofacial dysmorphology in mouse models of syndromic CS. These morphological trajectories increase our understanding of the progression of syndromic CS skull growth.

The role of the histone methyltransferase SET domain bifurcated 1 during palatal development.

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1fl/fl,Wnt1-Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1fl/fl,Wnt1-Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1fl/fl,Wnt1-Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1fl/fl,Wnt1-Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9.

Cell lineage- and expression-based inference of the roles of forkhead box transcription factor Foxc2 in craniofacial development.

BACKGROUND: Foxc2 is a member of the winged helix/forkhead (Fox) box family of transcription factors. Loss of function of Foxc2 causes craniofacial abnormalities such as cleft palate and deformed cranial base, but its role during craniofacial development remains to be elucidated. RESULTS: The contributions of Foxc2-positive and its descendant cells to the craniofacial structure at E18.5 were examined using a tamoxifen-inducible Cre driver mouse (Foxc2-CreERT2) crossed with the R26R-LacZ reporter mouse. Foxc2 expression at E8.5 is restricted to the cranial mesenchyme, contributing to specific components including the cranial base, sensory capsule, tongue, upper incisor, and middle ear. Expression at E10.5 was still positively regulated in most of those regions. In situ hybridization analysis of Foxc2 and its closely related gene, Foxc1, revealed that expression domains of these genes largely overlap in the cephalic mesenchyme. Meanwhile, the tongue expressed Foxc2 but not Foxc1, and its development was affected by the neural crest-specific deletion of Foxc2 in mice (Wnt1-Cre; Foxc2fl/fl ). CONCLUSIONS: Foxc2 is expressed in cranial mesenchyme that contributes to specific craniofacial tissue components from an early stage, and it seems to be involved in their development in cooperation with Foxc1. Foxc2 also has its own role in tongue development.

Heterozygous mutation of the splicing factor Sf3b4 affects development of the axial skeleton and forebrain in mouse.

BACKGROUND: Splicing factor 3B subunit 4 (SF3B4) is a causative gene of an acrofacial dysostosis, Nager syndrome. Although in vitro analyses of SF3B4 have proposed multiple noncanonical functions unrelated to splicing, less information is available based on in vivo studies using model animals. RESULTS: We performed expression and functional analyses of Sf3b4 in mice. The mouse Sf3b4 transcripts were found from two-cell stage, and were ubiquitously present during embryogenesis with high expression levels in several tissues such as forming craniofacial bones and brain. In contrast, expression of a pseudogene-like sequence of mouse Sf3b4 (Sf3b4_ps) found by in silico survey was not detected up to embryonic day 10. We generated a Sf3b4 knockout mouse using CRISPR-Cas9 system. The homozygous mutant mouse of Sf3b4 was embryonic lethal. The heterozygous mutant of Sf3b4 mouse (Sf3b4+/- ) exhibited smaller body size compared to the wild-type from postnatal to adult period, as well as homeotic posteriorization of the vertebral morphology and flattened calvaria. The flattened calvaria appears to be attributable to mild microcephaly due to a lower cell proliferation rate in the forebrain. CONCLUSIONS: Our study suggests that Sf3b4 controls anterior-posterior patterning of the axial skeleton and guarantees cell proliferation for forebrain development in mice.

Differing contributions of the first and second pharyngeal arches to tympanic membrane formation in the mouse and chick.

We have proposed that independent origins of the tympanic membrane (TM), consisting of the external auditory meatus (EAM) and first pharyngeal pouch, are linked with distinctive middle ear structures in terms of dorsal-ventral patterning of the pharyngeal arches during amniote evolution. However, previous studies have suggested that the first pharyngeal arch (PA1) is crucial for TM formation in both mouse and chick. In this study, we compare TM formation along the anterior-posterior axis in these animals using Hoxa2 expression as a marker of the second pharyngeal arch (PA2). In chick, the EAM begins to invaginate at the surface ectoderm of PA2, not at the first pharyngeal cleft, and the entire TM forms in PA2. Chick-quail chimera that have lost PA2 and duplicated PA1 suggest that TM formation is achieved by developmental interaction between a portion of the EAM and the columella auris in PA2, and that PA1 also contributes to formation of the remaining part of the EAM. By contrast, in mouse, TM formation is highly associated with an interdependent relationship between the EAM and tympanic ring in PA1.

Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors.

Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor ß2 (trß2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss- and gain-of-function experiments show that L-opsin expression requires trß2 activity before cone differentiation. Ectopic expression of trß2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trß2 expression could explain why some species have mixed, and others have pure, cone types.

The Dlx genes as clues to vertebrate genomics and craniofacial evolution.

The group of Dlx genes belongs to the homeobox-containing superfamily, and its members are involved in various morphogenetic processes. In vertebrate genomes, Dlx genes exist as multiple paralogues generated by tandem duplication followed by whole genome duplications. In this review, we provide an overview of the Dlx gene phylogeny with an emphasis on the chordate lineage. Referring to the Dlx gene repertoire, we discuss the establishment and conservation of the nested expression patterns of the Dlx genes in craniofacial development. Despite the accumulating genomic sequence resources in diverse vertebrates, embryological analyses of Dlx gene expression and function remain limited in terms of species diversity. By supplementing our original analysis of shark embryos with previous data from other osteichthyans, such as mice and zebrafish, we support the previous speculation that the nested Dlx expression in the pharyngeal arch is likely a shared feature among all the extant jawed vertebrates. Here, we highlight several hitherto unaddressed issues regarding the evolution and function of Dlx genes, with special reference to the craniofacial development of vertebrates.

On the homology of the shoulder girdle in turtles.

The shoulder girdle in turtles is encapsulated in the shell and has a triradiate morphology. Due to its unique configuration among amniotes, many theories have been proposed about the skeletal identities of the projections for the past two centuries. Although the dorsal ramus represents the scapular blade, the ventral two rami remain uncertain. In particular, the ventrorostral process has been compared to a clavicle, an acromion, and a procoracoid based on its morphology, its connectivity to the rest of the skeleton and to muscles, as well as with its ossification center, cell lineage, and gene expression. In making these comparisons, the shoulder girdle skeleton of anurans has often been used as a reference. This review traces the history of the debate on the homology of the shoulder girdle in turtles. And based on the integrative aspects of developmental biology, comparative morphology, and paleontology, we suggest acromion and procoracoid identities for the two ventral processes.

Development of the hyolaryngeal architecture in horseshoe bats: insights into the evolution of the pulse generation for laryngeal echolocation.

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.

Origin of the unique morphology of the shoulder girdle in turtles.

The shoulder girdle of turtles has a triradiate morphology. Although its dorsal process represents the scapular blade, the skeletal identities of the two ventral processes remain uncertain. To elucidate the question, developmental patterns of the girdles were compared between Chinese soft-shelled turtles, chickens, and mice. Despite the morphological diversity of adults, the initial primordia of the shoulder girdles showed similar morphological patterns. The ventral two processes developed from the anlagen comparable to those of the acromion and the coracoid in other amniotes. The developmental pattern of the acromion is very similar among embryos, whereas that of the coracoid in mammals differs from that in non-mammals, implying that coracoids are not homologous between non-mammals and mammals. Therefore, amniotes have retained the ancestral pattern of the girdle anlage, and the shoulder girdle of turtles has been achieved through a transformation of the pattern in the late ontogenic period.

An immunohistochemical study of matrix components in primary and secondary cartilages of embryonic chick skull.

OBJECTIVES: This study aimed to compare the extracellular matrix of primary cartilage with the secondary cartilage of chicks using immunohistochemical analyses in order to understand the features of chick secondary chondrogenesis. METHODS: Immunohistochemical analysis was performed on the extracellular matrix of quadrate (primary), squamosal, surangular, and anterior pterygoid secondary cartilages using various antibodies targeting the extracellular matrix of cartilage and bone. RESULTS: The localization of collagen types I, II, and X, versican, aggrecan, hyaluronan, link protein, and tenascin-C was identified in the quadrate cartilage, with variations within and between the regions. Newly formed squamosal and surangular secondary cartilages showed simultaneous immunoreactivity for all molecules investigated. However, collagen type X immunoreactivity was not observed, and there was weak immunoreactivity for versican and aggrecan in the anterior pterygoid secondary cartilage. CONCLUSIONS: The immunohistochemical localization of extracellular matrix in the quadrate (primary) cartilage was comparable to that of long bone (primary) cartilage in mammals. The fibrocartilaginous nature and rapid differentiation into hypertrophic chondrocytes, which are known structural features of secondary cartilage, were confirmed in the extracellular matrix of squamosal and surangular secondary cartilages. Furthermore, these tissues appear to undergo developmental processes similar to those in mammals. However, the anterior pterygoid secondary cartilage exhibited unique features that differed from primary and other secondary cartilages, suggesting it is formed through a distinct developmental process.

Anatomy and homology of the caudal auricular muscles in greater short-nosed fruit bat (Cynopterus sphinx).

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.

Augmented effect of fibroblast growth factor 18 in bone morphogenetic protein 2-induced calvarial bone healing by activation of CCL2/CCR2 axis on M2 macrophage polarization.

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.

Development of the head and trunk mesoderm in the dogfish, Scyliorhinus torazame: II. Comparison of gene expression between the head mesoderm and somites with reference to the origin of the vertebrate head.

The vertebrate mesoderm differs distinctly between the head and trunk, and the evolutionary origin of the head mesoderm remains enigmatic. Although the presence of somite-like segmentation in the head mesoderm of model animals is generally denied at molecular developmental levels, the appearance of head cavities in elasmobranch embryos has not been explained, and the possibility that they may represent vestigial head somites once present in an amphioxus-like ancestor has not been ruled out entirely. To examine whether the head cavities in the shark embryo exhibit any molecular signatures reminiscent of trunk somites, we isolated several developmentally key genes, including Pax1, Pax3, Pax7, Pax9, Myf5, Sonic hedgehog, and Patched2, which are involved in myogenic and chondrogenic differentiation in somites, and Pitx2, Tbx1, and Engrailed2, which are related to the patterning of the head mesoderm, from an elasmobranch species, Scyliorhinus torazame. Observation of the expression patterns of these genes revealed that most were expressed in patterns that resembled those found in amniote embryos. In addition, the head cavities did not exhibit an overt similarity to somites; that is, the similarity was no greater than that of the unsegmented head mesoderm in other vertebrates. Moreover, the shark head mesoderm showed an amniote-like somatic/visceral distinction according to the expression of Pitx2, Tbx1, and Engrailed2. We conclude that the head cavities do not represent a manifestation of ancestral head somites; rather, they are more likely to represent a derived trait obtained in the lineage of gnathostomes.

Rare coronary anastomoses between the aorta, pulmonary trunk, left coronary artery, and subclavian artery.

We report a rare case of coronary anastomoses in an 83-year-old male cadaveric heart. Anomalous vessels arose from the right sinus of the aorta, left main coronary artery, left anterior descending artery, left anterior medial atrial artery, and left subclavian artery. These vessels bifurcated and anastomosed, and finally connected to the pulmonary trunk. The present case is categorized as a multilateral coronary artery fistula in cardiology.

Temporal and regulatory dynamics of the inner ear transcriptome during development in mice.

The inner ear controls hearing and balance, while the temporal molecular signatures and transcriptional regulatory dynamics underlying its development are still unclear. In this study, we investigated time-series transcriptome in the mouse inner ear from embryonic day 11.5 (E11.5) to postnatal day 7 (P7) using bulk RNA-Seq. A total of 10,822 differentially expressed genes were identified between pairwise stages. We identified nine significant temporal expression profiles using time-series expression analysis. The constantly down-regulated profiles throughout the development are related to DNA activity and neurosensory development, while the constantly upregulated profiles are related to collagen and extracellular matrix. Further co-expression network analysis revealed that several hub genes, such as Pnoc, Cd9, and Krt27, are related to the neurosensory development, cell adhesion, and keratinization. We uncovered three important transcription regulatory paths during mice inner ear development. Transcription factors related to Hippo/TGFß signaling induced decreased expressions of genes related to the neurosensory and inner ear development, while a series of INF genes activated the expressions of genes in immunoregulation. In addition to deepening our understanding of the temporal and regulatory mechanisms of inner ear development, our transcriptomic data could fuel future multi-species comparative studies and elucidate the evolutionary trajectory of auditory development.

Evolution of oropharyngeal patterning mechanisms involving Dlx and endothelins in vertebrates.

In jawed vertebrates, the Dlx code, or nested expression patterns of Dlx genes, specify the dorsoventral polarity of pharyngeal arches, downstream of endothelin-1 (Edn-1) and its effectors, Bapx1 (Nkx3.2) and dHand (Hand2). To elucidate the evolution of the specification mechanism of the oropharyngeal skeletal system, lamprey homologs of Dlx, Edn, endothelin receptor (Ednr), Bapx1, and dHand were identified. Our analysis suggested that the Edn gene family emerged at the advent of vertebrates, and that gene duplications leading to the different Edn gnathostome subtypes (Edn1-3) occurred before the cyclostome-gnathostome split. This timing of gene duplications, giving rise to multiple subtypes, was also implied for Dlx, Ednr, Hand, and Bapx. In lamprey embryos, nested expressions of Dlx genes were not observed in pharyngeal arches, nor was any focal expression of Bapx1, known in gnathostomes to specify the jaw joint. The dHand homolog, however, was expressed more intensively ventrally, as in gnathostomes. Lamprey homologs of Edn-1 and EdnrA were also shown to be expressed as described in mice, indicating involvement of this signaling pathway in the craniofacial patterning early in vertebrate evolution. These results suggest that the last common ancestor of all the extant vertebrates would have possessed basic gene repertoires involved in oropharyngeal patterning in gnathostomes, but the elaborate genetic program leading to the Dlx code is likely to have been acquired uniquely in gnathostomes.

Overview of the transcriptome profiles identified in hagfish, shark, and bichir: current issues arising from some nonmodel vertebrate taxa.

Because of their crucial phylogenetic positions, hagfishes, sharks, and bichirs are recognized as key taxa in our understanding of vertebrate evolution. The expression patterns of the regulatory genes involved in developmental patterning have been analyzed in the context of evolutionary developmental studies. However, in a survey of public sequence databases, we found that the large-scale sequence data for these taxa are still limited. To address this deficit, we used conventional Sanger DNA sequencing and a next-generation sequencing technology based on 454 GS FLX sequencing to obtain expressed sequence tags (ESTs) of the Japanese inshore hagfish (Eptatretus burgeri; 161,482 ESTs), cloudy catshark (Scyliorhinus torazame; 165,819 ESTs), and gray bichir (Polypterus senegalus; 34,336 ESTs). We deposited the ESTs in a newly constructed database, designated the "Vertebrate TimeCapsule." The ESTs include sequences from genes that can be effectively used in evolutionary developmental studies; for instance, several encode cartilaginous extracellular matrix proteins, which are central to an understanding of the ways in which evolutionary processes affected the skeletal elements, whereas others encode regulatory genes involved in craniofacial development and early embryogenesis. Here, we discuss how hagfishes, sharks, and bichirs contribute to our understanding of vertebrate evolution, we review the current status of the publicly available sequence data for these three taxa, and we introduce our EST projects and newly developed database.