The autonomic nervous system, which includes the sympathetic neurons and adrenal medulla, originates from the neural crest. Combining avian blood vessel-specific gene manipulation and mouse genetics, we addressed a long-standing question of how neural crest cells (NCCs) generate sympathetic and medullary lineages during embryogenesis. We found that the dorsal aorta acts as a morphogenetic signaling center that coordinates NCC migration and cell lineage segregation. Bone morphogenetic proteins (BMPs) produced by the dorsal aorta are critical for the production of the chemokine stromal cell-derived factor-1 (SDF -1) and Neuregulin 1 in the para-aortic region, which act as chemoattractants for early migration. Later, BMP signaling is directly involved in the sympatho-medullary segregation. This study provides insights into the complex developmental signaling cascade that instructs one of the earliest events of neurovascular interactions guiding embryonic development.

The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off", works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes.

During early morphogenesis, tissue segregation is often accompanied by changes in cell shape. To understand how such coordination is regulated, somitogenesis was used as a model. When a somite forms in the anterior end of the presomitic mesoderm, an intersomitic boundary (gap) emerges, and it is rapidly followed by cell epithelialization at this border. It has been known that the gap formation is regulated by intercellular signals. We here demonstrate that cMeso-1, the chicken homolog of mouse Mesp2, upregulates EphA4 in the cells located posteriorly to a forming boundary. This in turn activates EphrinB2-reverse signals in the anteriorly juxtaposed cells, where the EphrinB2 signal is sufficient to cause a gap formation and cell epithelialization cell-autonomously. During these processes, Cdc42 needs to be repressed via tyrosine phosphorylation of EphrinB2. This is the first demonstration that Ephrin-reverse signal acts as a platform that couples distinct morphogenetic changes in cell polarity and tissue shape.Ephrin ͉ Epithelialization ͉ Segmentation

We studied, using avian embryos, mechanisms underlying the three-dimensional assembly of the dorsal aorta, the first-forming embryonic vessel in amniotes. This vessel originates from two distinct cell populations, the splanchnic and somitic mesoderms. We have unveiled a role for Notch signaling in the somitic contribution. Upon activation of Notch signaling, a subpopulation of cells in the posterior half of individual somites migrates ventrally toward the primary dorsal aorta of splanchnic origin. After reaching the primary aorta, these somitic cells differentiate into the definitive aortic endothelial cells. This Notch-induced ventral migration is mediated by Eph-rinB2 and by an attractant action of the primary aorta. Furthermore, long-term chasing of cells by transposon-mediated gene transfer reveals that the segmentally provided endothelial cells of somitic origin in the dorsal aorta ultimately populate the entire region of the vessel. We demonstrate the molecular and cellular mechanisms underlying the formation of embryonic blood vessels from mesenchymal cells.

Because of its lower sensitivity and accuracy, the non-lifting sign will not replace endoscopic assessment. If a lesion does not lift, this can make resection technically difficult, but does not reliably predict deeper cancerous invasion.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)‐containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide‐ and 5‐hydroxythalidomide‐induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell‐free protein synthesis system, PLZF was identified as a thalidomide‐dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5‐hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5‐hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide‐dependent CRBN substrate SALL4, was induced by thalidomide or 5‐hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide‐induced phenotypes. Our findings implicate PLZF as an important thalidomide‐induced CRBN neosubstrate involved in thalidomide teratogenicity.

Unlike microevolutionary processes, little is known about the genetic basis of macroevolutionary processes. One of these magnificent examples is the transition from non-avian dinosaurs to birds that has created numerous evolutionary innovations such as self-powered flight and its associated wings with flight feathers. By analysing 48 bird genomes, we identified millions of avian-specific highly conserved elements (ASHCEs) that predominantly (>99%) reside in non-coding regions. Many ASHCEs show differential histone modifications that may participate in regulation of limb development. Comparative embryonic gene expression analyses across tetrapod species suggest ASHCE-associated genes have unique roles in developing avian limbs. In particular, we demonstrate how the ASHCE driven avian-specific expression of gene Sim1 driven by ASHCE may be associated with the evolution and development of flight feathers. Together, these findings demonstrate regulatory roles of ASHCEs in the creation of avian-specific traits, and further highlight the importance of cis-regulatory rewiring during macroevolutionary changes.

The involvement of VEGF-A as well as the therapeutic efficacy of angiogenesis inhibitors in diabetic nephropathy have been reported. We recently reported the therapeutic effects of vasohibin-1 (VASH-1), an endogenous angiogenesis inhibitor, in a type 1 diabetic nephropathy model (Nasu T, Maeshima Y, Kinomura M, Hirokoshi-Kawahara K, Tanabe K, Sugiyama H, Sonoda H, Sato Y, Makino H. Diabetes 58: 2365-2375, 2009). In this study, we investigated the therapeutic efficacy of VASH-1 on renal alterations in obese mice with type 2 diabetes. Diabetic db/db mice received intravenous injections of adenoviral vectors encoding human VASH-1 (AdhVASH-1) and were euthanized 8 wk later. AdhVASH-1 treatment resulted in significant suppression of glomerular hypertrophy, glomerular hyperfiltration, albuminuria, increase in the CD31(+) glomerular endothelial area, F4/80(+) monocyte/macrophage infiltration, the accumulation of type IV collagen, and mesangial matrix. An increase in the renal levels of VEGF-A, VEGFR-2, transforming growth factor (TGF)-β1, and monocyte chemoattractant protein-1 in diabetic animals was significantly suppressed by AdhVASH-1 (immunoblotting). AdhVASH-1 treatment significantly recovered the loss and altered the distribution patterns of nephrin and zonula occludens (ZO)-1 and suppressed the increase in the number of fibroblast-specific protein-1 (FSP-1(+)) and desmin(+) podocytes in diabetic mice. In vitro, recombinant human VASH-1 (rhVASH-1) dose dependently suppressed the upregulation of VEGF induced by high ambient glucose (25 mM) in cultured mouse podocytes. In addition, rhVASH-1 significantly recovered the mRNA levels of nephrin and the protein levels of ZO-1 and P-cadherin and suppressed the increase in protein levels of desmin, FSP-1, Snail, and Slug in podocytes under high-glucose condition. Taken together, these results suggest the potential use of VASH-1 as a novel therapeutic agent in type 2 diabetic nephropathy mediated via antiangiogenic effects and maintenance of podocyte phenotype in association with antiproteinuric effects.