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.

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. K E Y W O R D Shomeotic posteriorization, microcephaly, Nager syndrome, splicing factor Takahiko Yamada and Masaki Takechi contributed equally to this study.

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 (Fgfr2 S252W/+ ) around 3 weeks old as seen in Crouzon syndrome model mice (Fgfr2c C342Y/+ ). 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 Fgfr2 S252W/+ and Fgfr2c C342Y/+ 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 cranial base is a structure mainly formed through endochondral ossification and integrated into the craniofacial complex, which acts as an underlying platform for the developing brain. Foxc1 is an indispensable regulator during intramembranous and endochondral ossification. In this study, we found that the spontaneous loss of Foxc1 function in a mouse (congenital hydrocephalous), Foxc1 , demonstrated the anterior cranial base defects, including unossified presphenoid and lack of middle part of the basisphenoid bone. Hypoplastic presphenoid primordial cartilage (basal portion of the trabecular cartilage [bTB]) and a lack of the middle part of basisphenoid primordial cartilage (the hypophyseal cartilage) were consistently observed at earlier developmental stage. Foxc1 was expressed robustly and ubiquitously in undifferentiated mesenchyme of the cranial base-forming area in E11.0 wild-type fetuses. Once chondrogenesis commenced, the expression was downregulated and later limited to the perichondrium. Detection of transcripts of Collagen type2 A1 (Col2a1) revealed that both bTB and the anterior part of the hypophyseal cartilage developing anterior to the persistent epithelial stalk of the anterior lobe of the pituitary gland were suppressed in the Foxc1 . Proliferation activity of chondrocyte precursor cells was higher in the Foxc1 . Loss of Foxc1 function only in the neural crest cell lineage (Wnt1-cre;Foxc1 ) showed ossification of the posterior part of the hypophyseal cartilage derived from the mesoderm. These findings suggest that Foxc1 is an important regulator to further chondrogenesis and initiate the ossification of the presphenoid and basisphenoid bones.

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 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.

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.

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.

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/TGFβ 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.