Developmental Morphology of the Head Mesoderm and Reevaluation of Segmental Theories of the Vertebrate Head: Evidence from Embryos of an Agnathan Vertebrate, Lampetra japonica

Due to the peculiar morphology of its preotic head, lampreys have long been treated as an intermediate animal which links amphioxus and gnathostomes. To reevaluate the segmental theory of classical comparative embryology, mesodermal development was observed in embryos of a lamprey, Lampetra japonica, by scanning electron microscopy and immunohistochemistry. Signs of segmentation are visible in future postotic somites at an early neurula stage, whereas the rostral mesoderm is unsegmented and rostromedially confluent with the prechordal plate. The premandibular and mandibular mesoderm develop from the prechordal plate in a caudal to rostral direction and can be called the preaxial mesoderm as opposed to the caudally developing gastral mesoderm. With the exception of the premandibular mesoderm, the head mesodermal sheet is secondarily regionalized by the otocyst and pharyngeal pouches into the mandibular mesoderm, hyoid mesoderm, and somite 0. The head mesodermal components never develop into cephalic myotomes, but the latter develop only from postotic somites. These results show that the lamprey embryo shows a typical vertebrate phylotype and that the basic mesodermal configuration of vertebrates already existed prior to the split of agnatha-gnathostomata; lamprey does not represent an intermediate state between amphioxus and gnathostomes. Unlike interpretations of theories of head segmentation that the mesodermal segments are primarily equivalent along the axis, there is no evidence in vertebrate embryos for the presence of preotic myotomes. We conclude that mesomere-based theories of head metamerism are inappropriate and that the formulated vertebrate head should possess the distinction between primarily unsegmented head mesoderm which includes preaxial components at least in part and somites in the trunk which are shared in all the known vertebrate embryos as the vertebrate phylotype.     

Function of Pax1 and Pax9 in the sclerotome of medaka fish

We examined the expression and functions of Pax1 and Pax9 in a teleost fish, the medaka Oryzias latipes. While Pax1 and Pax9 show distinct expression in the sclerotome in amniotes, we could not detect the differential expression of Pax1 and Pax9 in the developing sclerotome of the medaka. Furthermore, unlike the mouse, in which Pax1 is essential for development of the vertebral body, and where the neural arch is formed independent of either Pax1 or Pax9, our morpholino knockdown experiments revealed that both Pax1 and Pax9 are indispensable for the development of the vertebral body and neural arch. Therefore, we conclude that after gene duplication, Pax1 and Pax9 subfunctionalize their roles in the sclerotome independently in teleosts and amniotes. In Stage-30 embryo, Pax9 was strongly expressed in the posterior mesoderm, as was also observed for mouse Pax9. Since this expression was not detected for Pax1 in the mouse or fish, this new expression in the posterior mesoderm likely evolved in Pax9 of ancestral vertebrates after gene duplication. Two-month-old fish injected with Pax9 morpholino oligonucleotide showed abnormal morphology in the tail hypural skeletal element, which may have been related to this expression.     

叉头框-c2基因在主动脉弓发育过程中的作用

为了研究叉头框-c2（Forkhead Box c2, Fox c2）基因在心血管发生和发育中的作用, 通过制作小鼠的Fox c2 基因无效突变,解析该基因缺失鼠主动脉弓的异常发育状况.纯合子胎鼠从12.5天胚胎(embryo, E)开始有宫内死亡;即使完成宫内发育过程,新生鼠出生24 h后也全部死亡.这些鼠全部表现出与人的先天性心血管发育缺陷相似的B型或C型主动脉弓离断.杂合子鼠发育正常.E10.5胚胎的原位杂交分析显示,Fox c2 mRNA在第三、第四和第六弓型动脉强烈表达,而第四弓型动脉在E10.5胚胎后逐渐消失.这些结果表明,在主动脉弓形成过程中,Fox c2基因产物是左第四弓形动脉广泛改建所必需.     

An in vitro and finite element study of load redistribution in the midfoot

A good knowledge of midfoot biomechanics is important in understanding the biomechanics of the entire foot,but it has never been investigated thoroughly in the literature.This study carried out in vitro experiments and finite element analysis to investigate the midfoot biomechanics.A foot-ankle finite element model simulating the mid-stance phase of the normal gait was developed and the model validated in in vitro experimental tests.Experiments used seven in vitro samples of fresh human cadavers.The simulation found that the first principal stress peaks of all midfoot bones occurred at the navicular bone and that the tensile force of the spring ligament was greater than that of any other ligament.The experiments showed that the longitudinal strain acting on the medial cuneiform bone was-26.2±10.8μ-strain,and the navicular strain was-240.0±169.1μ-strain along the longitudinal direction and 65.1±25.8μ-strain along the transverse direction.The anatomical position and the spring ligament both result in higher shear stress in the navicular bone.The load from the ankle joint to five branches of the forefoot is redistributed among the cuneiforms and cuboid bones.Further studies on the mechanism of loading redistribution will be helpful in understanding the biomechanics of the entire foot.     

<Emphasis Type="Italic">Hoxa3</Emphasis> regulates the proliferation and differentiation of the third pharyngeal arch mesenchyme in mice

Genetic disruption of Hoxa3 results in bilateral defects of the common carotid artery, which is derived from the third branchial arch artery. The tunica media of the great arteries derived from the arch arteries is formed by the ectomesenchymal neural crest cells. To examine the etiology of the regression of the third arch artery, we generated Hoxa3 homozygous null mutant embryos that expressed a lacZ marker transgene driven by a connexin43 (Cx43): promoter in the neural crest cells. The expression of -galactosidase in these mouse embryos was examined by both whole-mount X-gal staining and immunohistochemistry with the monoclonal -galactosidase antibody on sections. The migration of neural crest cells from the neural tube to the third branchial arch was not affected in the Hoxa3 homozygotes. The initial formation of the third arch artery was also not disturbed. The artery, however, regressed at embryonic day 11.5 (E11.5), when differentiation of the third pharyngeal arch began. The internal and external carotid arteries arose from the dorsal aorta in E12.5 null mutants, which showed an abnormal persistence of the ductus caroticus. The third pharyngeal arch of wild-type mice fuses with the fourth and second arches at E12.0. In the Hoxa3 null mutants, however, the fusion was delayed, and the hypoplastic third pharyngeal arch was still discerned at E12.5. Moreover, the number of proliferating cells in the third arch of the null mutants was small compared with that in the wild-type. Thus, Hoxa3 is required for the growth and differentiation of the third pharyngeal arch. The defective development of the third pharyngeal arch may induce the anomalies of the carotid artery system. This work was supported in part by a grant (no. 14570026) from the Ministry of Education of Japan to Y.K.     

Comparison of the effects of HGF,BDNF, CT‐1, CNTF,and the branchial arches on the growth of embryonic cranial motor neurons

In the developing embryo, axon growth and guidance depend on cues that include diffusible molecules. We have shown previously that the branchial arches and hepatocyte growth factor (HGF) are growth‐promoting and chemoattractant for young embryonic cranial motor axons. HGF is produced in the branchial arches of the embryo, but a number of lines of evidence suggest that HGF is unlikely to be the only factor involved in the growth and guidance of these axons. Here we investigate whether other neurotrophic factors could be involved in the growth of young cranial motor neurons in explant cultures. We find that brain‐derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and cardiotrophin‐1 (CT‐1) all promote the outgrowth of embryonic cranial motor neurons, while glial cell line‐derived neurotrophic factor (GDNF) and neurotrophin‐3 (NT‐3) fail to affect outgrowth. We next examined whether HGF and the branchial arches had similar effects on motor neuron subpopulations at different axial levels. Our results show that HGF acts as a generalized rather than a specific neurotrophic factor and guidance cue for cranial motor neurons. Although the branchial arches also had general growth‐promoting effects on all motor neuron subpopulations, they chemoattracted different axial levels differentially, with motor neurons from the caudal hindbrain showing the most striking response. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 101–114, 2002     

A three‐dimensional placoderm (stem‐group gnathostome) pharyngeal skeleton and its implications for primitive gnathostome pharyngeal architecture

The pharyngeal skeleton is a key vertebrate anatomical system in debates on the origin of jaws and gnathostome (jawed vertebrate) feeding. Furthermore, it offers considerable potential as a source of phylogenetic data. Well‐preserved examples of pharyngeal skeletons from stem‐group gnathostomes remain poorly known. Here, we describe an articulated, nearly complete pharyngeal skeleton in an Early Devonian placoderm fish, Paraplesiobatis heinrichsi Broili, from Hunsrück Slate of Germany. Using synchrotron light tomography, we resolve and reconstruct the three‐dimensional gill arch architecture of Paraplesiobatis and compare it with other gnathostomes. The preserved pharyngeal skeleton comprises elements of the hyoid arch (probable ceratohyal) and a series of branchial arches. Limited resolution in the tomography scan causes some uncertainty in interpreting the exact number of arches preserved. However, at least four branchial arches are present. The final and penultimate arches are connected as in osteichthyans. A single median basihyal is present as in chondrichthyans. No dorsal (epibranchial or pharyngobranchial) elements are observed. The structure of the pharyngeal skeleton of Paraplesiobatis agrees well with Pseudopetalichthys from the same deposit, allowing an alternative interpretation of the latter taxon. The phylogenetic significance of Paraplesiobatis is considered. A median basihyal is likely an ancestral gnathostome character, probably with some connection to both the hyoid and the first branchial arch pair. Unpaired basibranchial bones may be independently derived in chondrichthyans and osteichthyans.     

Retinoic acid affects craniofacial patterning by changing Fgf8 expression in the pharyngeal ectoderm

Retinoic acid signaling plays important roles in establishing normal patterning and cellular differentiation during embryonic development. In this study, we show that single administration of retinoic acid at embryonic day 8.5 causes homeotic transformation of the lower jaw into upper jaw-like structures. This homeosis was preceded by downregulation of Fgf8 and Sprouty expression in the proximal domain of the first pharyngeal arch. Downregulation of mesenchymal genes such as Dlx5, Hand2, Tbx1 and Pitx2 was also observed. The oropharynx in retinoic acid-treated embryos was severely constricted. Consistent with this observation, Patched expression in the arch endoderm and mesenchyme was downregulated. Thus, retinoic acid affects the expression of subsets of epithelial and mesenchymal genes, possibly disrupting the regional identity of the pharyngeal arch.     

Anti-apoptotic gene Bcl2 is required for stapes development and hearing

In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2^−/− mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2^−/− mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2^−/−Bim^−/− mice and partially rescued in Bcl2^−/−Bim^+/− mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2^−/− defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development.