Toshiyuki Yamagishi

Tenascin C may regulate the recruitment of smooth muscle cells during coronary artery development

Tenascin C (TNC) is an extracellular glycoprotein that is thought to be involved in tissue remodeling during organogenesis and regeneration. Using avian embryonic hearts, we investigated the spatiotemporal expression patterns of TNC during the formation of the proximal coronary artery. Immunohistochemistry showed that TNC was deposited around the developing coronary stem and that TNC colocalized with vascular smooth muscle α-actin. A quail-chick chimera, in which a quail proepicardial organ (PEO) had been transplanted, showed that quail tissue-derived cells contributed to the establishment of the endothelial and mural cells of the proximal coronary artery, and the quail tissue-derived mural cells displayed TNC. Proepicardial cells cultured in TNC showed the myofibroblast/smooth muscle cell phenotype and neutralizing anti-TNC antibody suppressed the expression of smooth muscle markers. These observations suggest that TNC plays a role in the mural smooth muscle development of the nascent proximal coronary artery.

Nodal signal is required for morphogenetic movements of epiblast layer in the pre-streak chick blastoderm

During axis formation in amniotes, posterior and lateral epiblast cells in the area pellucida undergo a counter‐rotating movement along the midline to form primitive streak (Polonaise movements). Using chick blastoderms, we investigated the signaling involved in this cellular movement in epithelial‐epiblast. In cultured posterior blastoderm explants from stage X to XI embryos, either Lefty1 or Cerberus‐S inhibited initial migration of the explants on chamber slides. In vivo analysis showed that inhibition of Nodal signaling by Lefty1 affected the movement of DiI‐marked epiblast cells prior to the formation of primitive streak. In Lefty1‐treated embryos without a primitive streak, Brachyury expression showed a patchy distribution. However, SU5402 did not affect the movement of DiI‐marked epiblast cells. Multi‐cellular rosette, which is thought to be involved in epithelial morphogenesis, was found predominantly in the posterior half of the epiblast, and Lefty1 inhibited the formation of rosettes. Three‐dimensional reconstruction showed two types of rosette, one with a protruding cell, the other with a ventral hollow. Our results suggest that Nodal signaling may have a pivotal role in the morphogenetic movements of epithelial epiblast including Polonaise movements and formation of multi‐cellular rosette.

In Vivo Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.

Epicardium is required for sarcomeric maturation and cardiomyocyte growth in the ventricular compact layer mediated by transforming growth factor β and fibroblast growth factor before the onset of coronary circulation

The epicardium, which is derived from the proepicardial organ (PE) as the third epithelial layer of the developing heart, is crucial for ventricular morphogenesis. An epicardial deficiency leads to a thin compact layer for the developing ventricle; however, the mechanisms leading to the impaired development of the compact layer are not well understood. Using chick embryonic hearts, we produced epicardium‐deficient hearts by surgical ablation or blockade of the migration of PE and examined the mechanisms underlying a thin compact myocardium. Sarcomeric maturation (distance between Z‐lines) and cardiomyocyte growth (size) were affected in the thin compact myocardium of epicardium‐deficient ventricles, in which the amounts of phospho‐smad2 and phospho‐ERK as well as expression of transforming growth factor (TGF)β2 and fibroblast growth factor (FGF)2 were reduced. TGFβ and FGF were required for the maturation of sarcomeres and growth of cardiomyocytes in cultured ventricles. In ovo co‐transfection of dominant negative (dN)‐Alk5 (dN‐TGFβ receptor I) and dN‐FGF receptor 1 to ventricles caused a thin compact myocardium. Our results suggest that immature sarcomeres and small cardiomyocytes are the causative architectures of an epicardium‐deficient thin compact layer and also that epicardium‐dependent signaling mediated by TGFβ and FGF plays a role in the development of the ventricular compact layer before the onset of coronary circulation.

Development of the dorsal ramus of the spinal nerve in the mouse embryo: Involvement of semaphorin 3A in dorsal muscle innervation

The spinal nerve, which is composed of dorsal root ganglion (DRG) sensory axons and spinal motor axons, forms the dorsal ramus projecting to the dorsal musculature. By using the free‐floating immunohistochemistry method, we closely examined the spatiotemporal pattern of the formation of the dorsal ramus and the relationship between its projection to the myotome/dorsal musculature and semaphorin 3A (Sema3A), which is an axonal guidance molecule. In embryonic day (E) 10.5–E11.5 wild‐type mouse embryos, we clearly showed the existence of a waiting period for the dorsal ramus projection to the myotome. In contrast, in E10.5–E11.5 Sema3A‐deficient embryos, the dorsal ramus fibers projected beyond the edge of the myotome without exhibiting the waiting period for projection. These results strongly suggest that the delayed innervation by dorsal ramus fibers may be caused by Sema3A‐induced axon repulsion derived from the myotome. Next, by performing culture experiments, we confirmed that E12.5 mouse axons responded to Sema3A‐induced repulsion. Together, our results imply that Sema3A may play a key role in the proper development of the dorsal ramus projection.

Local ventilation system successfully reduced formaldehyde exposure during gross anatomy dissection classes.

Dear Editor, In the recent Acta Anatomica Nipponica (Kaibogaku Zasshi Vol. 85, No. 1, 2010)—a special issue concerning the improvement of the formaldehyde (FA) environment in gross-anatomy laboratories—four papers were published, two of which described originally developed dissection tables equipped with local ventilation systems (Shinoda and Oba 2010; Kikuta et al. 2010) and two of which outlined the environmental health hazards caused by FA (Uchiyama 2010; Sakamoto and Miyake 2010). Due to its effectiveness and low cost, the FA solution is widely used in Japanese medical and dental schools to embalm human bodies donated for use in gross anatomy dissection classes. However, the gaseous FA that evaporates from embalmed bodies causes not only acute irritation to the eyes and respiratory tract but also chronic non-threshold carcinogenicity. According to a risk-based evaluation of FA, the Ministry of Health, Labor, and Welfare in Japan has set the administrative level of FA to 0.1 ppm in working environments in which FA is handled. In our medical school, the dissection laboratory for students (L23 9 W12 9 H3.3 m, in which 20–23 bodies prepared with 10 l of 5.5% FA/30% ethanol solution are used) had a high-performance general (whole-room) ventilation system with a competence of 25000 m/h; however, the mean FA concentration of the room (‘‘A’’ measurement) and the estimated maximum exposure to FA (‘‘B’’ measurement) during dissection classes were 0.520 and 0.480 ppm, respectively (HPLC analysis performed by Panasonic Health Organization Science Center of Industrial Hygiene, Osaka, Japan). To reduce FA exposure during dissection classes, we introduced 23 dissection tables with local ventilation apparatus (Meiko Medical, Fukuoka, Japan). The details of this system have already been described elsewhere (Shinoda and Oba 2010). Briefly, the system consists of a simple plenum-chambered dissection table and a transparent vinyl duct which connects the table to the pre-existing general ventilation duct via a flow control valve in the ceiling. The 40 pre-existing, randomly oriented air-supply openings in the ceiling were not replaced (no downward flow of air for each dissecting table). The total ventilation flow rate was 18 m/min/table. The local ventilation system we introduced successfully reduced the FA concentration of the room during dissection classes. The A and B FA measurements were 0.035 and 0.054 ppm, respectively. Using a photoelectronic method (FP-30, Rikenkeiki, Tokyo, Japan), we also measured the 30-min mean FA concentration at the center of the dissection room (1.2 m above the floor) and at the corner of a dissection table (0.5 m above the table) in every class 30–60 min after the start of the dissection (Fig. 1). The mean FA concentrations at the center of the room and the corner of the table were 0.056 (n = 41) and 0.057 ppm (n = 41), respectively. FA concentrations higher than 0.1 ppm were recorded for dissection schedules #8 and 9, during which the upper extremities were placed in the abducent position; thus, the source of the FA was out of the effective range of the local ventilation system. Thirty-nine of the 41 measurements of FA concentration at the center of the room and 40 of the 41 measurements at the corner of M. Takahashi M. Abe T. Yamagishi K. Nakatani Y. Nakajima (&) Anatomy and Cell Biology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan e-mail: yuji@med.osaka-cu.ac.jp

Myocardial progenitors in the pharyngeal regions migrate to distinct conotruncal regions

Background: The cardiac progenitor cells for the outflow tract (OFT) reside in the visceral mesoderm and mesodermal core of the pharyngeal region, which are defined as the secondary and anterior heart fields (SHF and AHF), respectively. Results: Using chick embryos, we injected fluorescent‐dye into the SHF or AHF at stage 14, and the destinations of the labeled cells were examined at stage 31. Labeled cells from the right SHF were found in the myocardium on the left dorsal side of the OFT, and cells from the left SHF were detected on the right ventral side of the OFT. Labeled cells from the right and left AHF migrated to regions of the ventral wall of the OFT close to the aortic and pulmonary valves, respectively. Conclusion: These observations indicate that myocardial progenitors from the SHF and AHF contribute to distinct conotruncal regions and that cells from the SHF migrate rotationally while cells from the AHF migrate in a non‐rotational manner. Developmental Dynamics 241:284–293, 2012.

Expression of the Tgfβ2 Gene During Chick Embryogenesis

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) β2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49–92) using in situ hybridization. During cardiogenesis, Tgfβ2 was expressed in the endothelial/mesenchymal cells of the valvulo‐septal endocardial cushion tissue and in the epicardium until the end of embryogenesis. During the formation of major arteries, Tgfβ2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfβ2 was expressed in the mesenchymal cells in the presumptive limb regions at stage 16, and thereafter it was localized in the skeletal muscle progenitors. In addition, strong Tgfβ2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfβ2 mRNA was also detected in other mesoderm‐derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfβ2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfβ2 was expressed in the developing gut epithelium. Our results suggest that TGFβ2 plays an important role not only in epithelial‐mesenchymal interactions but also in cell differentiation and migration and cell death during chick embryogenesis. We also found that chick and mouse Tgfβ2 RNA show very similar patterns of expression during embryogenesis. Chick embryos can serve as a useful model to increase our understanding in the roles of TGFβ2 in cell–cell interactions, cell differentiation, and proliferation during organogenesis. Anat Rec, 2012.

Follistatin-like 5 is expressed in restricted areas of the adult mouse brain: Implications for its function in the olfactory system.

Follistatin‐like 5 (Fstl5), a member of the follistatin family of genes, encodes a secretory glycoprotein. Previous studies revealed that other members of this family including Fstl1 and Fstl3 play an essential role in development, homeostasis, and congenital disorders. However, the in vivo function of Fstl5 is poorly understood. To gain insight into the function of Fstl5 in the mouse central nervous system, we examined the Fstl5 expression pattern in the adult mouse brain. The results of in situ hybridization analysis showed a highly restricted pattern of Fstl5, namely, with localization in the olfactory system, hippocampal CA3 area and granular cell layer of the cerebellum. Restricted expression in the olfactory system suggests a possible role for Fstl5 in maintaining odor perception.

Impaired Development of Left Anterior Heart Field by Ectopic Retinoic Acid Causes Transposition of the Great Arteries

Background Transposition of the great arteries is one of the most commonly diagnosed conotruncal heart defects at birth, but its etiology is largely unknown. The anterior heart field (AHF) that resides in the anterior pharyngeal arches contributes to conotruncal development, during which heart progenitors that originated from the left and right AHF migrate to form distinct conotruncal regions. The aim of this study is to identify abnormal AHF development that causes the morphology of transposition of the great arteries. Methods and Results We placed a retinoic acid–soaked bead on the left or the right or on both sides of the AHF of stage 12 to 14 chick embryos and examined the conotruncal heart defect at stage 34. Transposition of the great arteries was diagnosed at high incidence in embryos for which a retinoic acid–soaked bead had been placed in the left AHF at stage 12. Fluorescent dye tracing showed that AHF exposed to retinoic acid failed to contribute to conotruncus development. FGF8 and Isl1 expression were downregulated in retinoic acid–exposed AHF, and differentiation and expansion of cardiomyocytes were suppressed in cultured AHF in medium supplemented with retinoic acid. Conclusions The left AHF at the early looped heart stage, corresponding to Carnegie stages 10 to 11 (28 to 29 days after fertilization) in human embryos, is the region of the impediment that causes the morphology of transposition of the great arteries.