Msx1 upregulates p27 expression to control cellular proliferation during valvuloseptal endocardial cushion formation in the chick embryonic heart.

Cushion tissues, the primordia of valves and septa of the adult heart, are formed in the atrioventricular (AV) and outflow tract (OFT) regions of the embryonic heart. The cushion tissues are generated by the endothelial-mesenchymal transition (EMT), involving many soluble factors, extracellular matrix, and transcription factors. Moreover, neural crest-derived mesenchymal cells also migrate into the OFT cushion. The transcription factor Msx1 is known to be expressed in the endothelial and mesenchymal cells during cushion tissue formation. However, its exact role in EMT during cushion tissue formation is still unknown. In this study, we investigated the expression patterns of Msx1 mRNA and protein during chick heart development. Msx1 mRNA was localized in endothelial cells of the AV region at Stage 14, and its protein was first detected at Stage 15. Thereafter, Msx1 mRNA and protein were observed in the endothelial and mesenchymal cells of the OFT and AV regions. in vitro assays showed that ectopic Msx1 expression in endothelial cells induced p27, a cell-cycle inhibitor, expression and inhibited fibroblast growth factor 4 (FGF4)-induced cell proliferation. Although the FGF signal reduced the EMT-inducing activities of transforming growth factor ß (TGFß), ectopic Msx1 expression in endothelial cells enhanced TGFß signaling-induced αSMA, an EMT marker, expression. These results suggest that Msx1 may support the transformation of endothelial cells due to a TGFß signal in EMT during cushion tissue formation.

Hypoxia-induced downregulation of Sema3a and CXCL12/CXCR4 regulate the formation of the coronary artery stem at the proper site.

BACKGROUND: During the formation of the coronary artery stem, endothelial strands from the endothelial progenitor pool surrounding the conotruncus penetrate into the aortic wall. Vascular endothelial growth factors (VEGFs) as well as CXCL12/CXCR4 signaling are thought to play a role in the formation of the coronary stem. However, the mechanisms regulating how endothelial strands exclusively invade into the aorta remain unknown. METHODS AND RESULTS: Immunohistochemistry showed that before the formation of endothelial strands, Sema3a was highly expressed in endothelial progenitors surrounding the great arteries. At the onset of/during invasion of endothelial strands into the aorta, Sema3a was downregulated and CXCR4 was upregulated in the endothelial strands. In situ hybridization showed that Cxcl12 was highly expressed in the aortic wall compared with in the pulmonary artery. Using avian embryonic hearts, we established two types of endothelial penetration assay, in which coronary endothelial strands preferentially invaded into the aorta in culture. Sema3a blocking peptide induced an excess number of endothelial strands penetrating into the pulmonary artery, whereas recombinant Sema3a inhibited the formation of endothelial strands. In cultured coronary endothelial progenitors, recombinant VEGF protein induced CXCR4-positive endothelial strands, which were capable of being attracted by CXCL12-impregnated beads. Monoazo rhodamine detected that hypoxia was predominant in aortic/subaortic region in ovo and hypoxic condition downregulated the expression of Sema3a in culture. CONCLUSION: Results suggested that hypoxia in the aortic region downregulates the expression of Sema3a, thereby enhancing VEGF activity to induce the formation of CXCR4-positive endothelial strands, which are subsequently attracted into the Cxcl12-positive aortic wall to connect the aortic lumen.

A novel case of multiple variations in the brachial plexus with the middle trunk originating from the C7 and C8.

The brachial plexus is an important nervous structure from which all major nerves to the upper limb arise. It typically originates from the anterior rami of the C5-T1 spinal nerves. As it passes laterally, the roots are successively organized into three trunks, six divisions, and three cords. The BP is susceptible to injury during the perinatal and postnatal periods, as well as in adulthood. Its structure can show considerable variation, and there is a wealth of literature describing its variations, providing indispensable information to neurosurgeons. Here, we report a novel unilateral variant of the brachial plexus found in an adult Japanese male cadaver. In this case, the middle trunk arose from the C7 and C8 spinal nerves, and the inferior trunk continued from the T1 alone. At the interscalene triangle, the subclavian artery was situated between the C8 and T1 nerves. The posterior cord arose from the posterior divisions of the superior and middle trunks, while the root from the T1 nerve/inferior trunk was absent. The anterior division of the middle trunk gave independent roots to the musculocutaneous and median nerves, without completely establishing the lateral cord. A communicating branch arose from the musculocutaneous nerve to join the median nerve. Some branches from the roots and cords also deviated from typical configurations. This case represents a rare combination of variations in the trunks, divisions, cords, and the median nerve and offers a valuable addition to the literature regarding variations in the brachial plexus.

Avian coronary endothelium is a mosaic of sinus venosus- and ventricle-derived endothelial cells in a region-specific manner.

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein-Tol2 system and fluorescent dye labeling, four types of quail-chick chimeras were made and quail-specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium-derived inner cells and the other was surface mesothelium-derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region-specific manner, and the sources of ECs were basically the same as those observed in mice.

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.

Epicardium is required for sarcomeric maturation and cardiomyocyte growth in the ventricular compact layer mediated by transforming growth factor ß 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)ß2 and fibroblast growth factor (FGF)2 were reduced. TGFß 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-TGFß 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 TGFß and FGF plays a role in the development of the ventricular compact layer before the onset of coronary circulation.

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.

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.

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.

Spatiotemporal patterns of the Huntingtin-interacting protein 1-related gene in the mouse head.

Huntingtin-interacting protein 1-related (Hip1r) was originally identified due to its homology to Huntingtin-interacting protein 1, which contributes to the development of Huntington's disease (HD). We studied the expression of the mouse Hip1r (mHip1r) gene in the mouse head by in situ hybridization. In early embryogenesis at embryonic day (E) 13, mHip1r expression was especially prominent in the olfactory epithelium, cerebral cortex layer 1, cortical plate, and dentate gyrus. During later development from E15 to E17, strong expression of mHip1r transcripts continued to be observed in the olfactory epithelium, cortical plate, and dentate gyrus. Furthermore, not only the subplate and subventricular zone of the cortex, but also secretory glands, such as the nasal gland and the submandibular gland, were mHip1r-positive. Other positive tissues included the retinal ganglion cells, vomeronasal organ, trigeminal ganglion, and the developing molar tooth. In the adult mouse brain, similar expression patterns were observed in the cerebral cortex layers and other brain regions except the cerebellum. Additionally, by using an antibody against mHip1r, we confirmed these expression patterns at the protein level. Specific expression of mHip1r in the embryonic brain and secretory glands suggests a possible role for Hip1r in normal development and in the pathology of HD.

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.

Expression of the Tgfß2 gene during chick embryogenesis.

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) ß2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49-92) using in situ hybridization. During cardiogenesis, Tgfß2 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, Tgfß2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfß2 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 Tgfß2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfß2 mRNA was also detected in other mesoderm-derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfß2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfß2 was expressed in the developing gut epithelium. Our results suggest that TGFß2 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 Tgfß2 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 TGFß2 in cell-cell interactions, cell differentiation, and proliferation during organogenesis.

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.

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.

Roles of TGFbeta and BMP during valvulo-septal endocardial cushion formation.

The primordia of valves and the atrioventricular septum arise from endocardial cushion tissue that is formed in the outflow tract (OFT) and in the atrioventricular (AV) regions during cardiogenesis. Abnormal development of the endocardial cushion results in various congenital heart diseases. Endocardial epithelial-mesenchymal transformation (EMT) is a critical process in cushion tissue formation and is regulated by many factors, such as growth factors, intercellular signaling molecules, transcription factors, and extracellular matrices. A signal that is produced by the myocardium of the AV and OFT regions and transferred to the adjacent endocardium across the extracellular matrix mediates EMT. Studies in vitro and genetic analyses have shown that transforming growth factor beta and bone morphogenetic protein play central roles in the regulation of EMT during cushion tissue formation.

Heart development before beating.

During heart development at the pregastrula stage, prospective heart cells reside in the posterior lateral region of the epiblast layer. Interaction of tissues between the posterior epiblast and hypoblast is necessary to generate the future heart mesoderm. Signaling regulating the interaction involves fibroblast growth factor (FGF)-8, Nodal, bone morphogenetic protein (BMP)-antagonist, and canonical Wnt and acts on the posterior epiblast to induce the expression of genes specific for the anterior lateral mesoderm. At the early gastrula stage, prospective heart cells accumulate at the posterior midline and migrate to the anterior region of the primitive streak. During gastrulation, future heart cells leave the primitive streak and migrate anterolaterally to form the left and right anterior lateral plate mesoderm including the precardiac mesoderm. At this stage, prospective heart cells receive endoderm-derived signals, including BMP, FGF, and Wnt-antagonist, and thereby become committed to the heart lineage. At the neurula stage, the left and right precardiac mesoderm move to the ventral midline and fuse, resulting in the formation of a single primitive heart tube. Therefore, a two-step signaling cascade, which includes tissue interaction between epiblast and hypoblast at the blastula stage and endoderm-derived signals during gastrulation, is required to generate a beating heart.

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.

Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was approximately 100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only approximately 1% with other gene delivery methods. Furthermore, in combination with negative selection, approximately 45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells.

ROCK1 expression is regulated by TGFbeta3 and ALK2 during valvuloseptal endocardial cushion formation.

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial-mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor-beta (TGFbeta), bone morphogenetic protein (BMP), and Rho-ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGFbeta or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up-regulated in endothelial/mesenchymal cells. A three-dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co-cultured with associated myocardium, ROCK1 expression was inhibited by either anti-TGFbeta3 antibody, anti-ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGFbeta3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGFbeta3 plus anti-ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up-regulated at the onset of EMT and that signaling mediated by TGFbeta3/ALK2 together with BMP is involved in the expression of ROCK1.

Induction of initial heart alpha-actin, smooth muscle alpha-actin, in chick pregastrula epiblast: the role of hypoblast and fibroblast growth factor-8.

During heart development at the gastrula stage, inhibition of bone morphogenetic protein (BMP) activity affects the heart specification but does not impair the expression of smooth muscle alpha-actin (SMA), which is first expressed in the heart mesoderm and recruited into initial heart myofibrils. Interaction of tissues between posterior epiblast and hypoblast at the early blastula stage is necessary to induce the expression of SMA, in which Nodal and Chordin are thought to be involved. Here we investigated the role of fibroblast growth factor-8 (FGF8) in the expression of SMA. In situ hybridization and reverse transcription-polymerase chain reaction showed that Fgf8b is expressed predominantly in the nascent hypoblast. Anti-FGF8b antibody inhibited the expression of SMA, cTNT, and Tbx5, which are BMP-independent heart mesoderm/early cardiomyocyte genes, but not Brachyury in cultured posterior blastoderm, and combined FGF8b and Nodal, but neither factor alone induced the expression of SMA in association with heart specific markers in cultured epiblast. Although FGF8b did not induce the upregulation of phospho-Smad2, anti-FGF8b properties suppressed phospho-Smad2 in cultured blastoderm. FGF8b was able to reverse the BMP-induced inhibition of cardiomyogenesis. The results suggest that FGF8b acts on the epiblast synergistically with Nodal at the pregastrula stage and may play a role in the expression of SMA during early cardiogenesis.