Ventral ectodermal ridge and ventral ectodermal groove: two distinct morphological features in the developing rat embryo tail

The ventral ectodermal ridge (VER) is a thickening of the surface ectoderm on the ventral side of the embryonic tail which resembles the apical ectodermal ridge of the limb bud. The morphological characteristics of the ventral part of the embryo tail were investigated in 10.5- to 14-day rat embryos by light microscopy of serial semithin sections and by scanning and transmission electron microscopy. In 10.5- to 11.5-day embryos the thickening of the ventral surface ectoderm includes the complete ventral midline of the tail and can be divided into two parts. The posterior part is elevated and represents the ventral ectodermal ridge. The anterior part is, in contrast to the ridge, concave, and we have termed it the ventral ectodermal groove (VEG). The cloacal membrane is located at its anterior end. Contacts between the VER and the mesenchymal cells are visible until an intact basal lamina is formed at 11.5 days. Similarly, the VEG is connected by elongated cell processes with the ventral part of the tail gut. Gap junctions are present between the apical parts of ridge and groove cells. The VEG flattens and disappears in 12-day embryos. At this stage the ridge is at its maximum height, simultaneously undergoing extensive cell death. The VER is no longer visible in 14-day rat embryos.     

Comparison of platelet-rich plasma,bovine BMP,and rhBMP-4 on bone matrix protein expression in vitro

This study investigated the potential use of platelet-rich plasma (PRP) in conjunction with mRNA expression of bone matrix proteins using bioassay and RT-PCR comparing bovine bone morphogenetic proteins (BMP), recombinant human BMP-4 (rhBMP-4) during rat bone marrow stromal cell (Mesenchymal Stem Cell) differentiation at 14 days. The results showed that all three growth factors were associated with significantly elevated alkaline phosphatase activity. PRP and bovine BMP resulted in increased protein content. The mRNA of type I collagen was expressed with all three growth factors and remained consistently elevated. Osteopontin was observed with PRP from days 1 to 7; bone sialoprotein expression was detected on days 1 and 3. PRP, bovine BMP and rhBMP-4 enhanced the steady-state expression of PDGF-A as time-dependent to day 14 and in PRP was the strongest. PTHr was expressed at days 1 and 5. Vascular endothelial growth factor expression was the most highly expressed after day 3. These findings suggest that PRP increases mRNA expression of bone matrix protein, enchances osteogenesis and angiogenesis in vitro.     

Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse

Background: Skeletal myogenesis is extensively influenced by the surrounding environment. However, how the extracellular matrix (ECM) affects morphogenesis of muscles is not well understood. Results: We mapped the three‐dimensional (3D) organization of fibronectin, tenascin, and laminin by immunofluorescence during early epaxial myogenesis in mouse embryos. We define four stages of dermomyotome/myotome development and reveal the 3D organization of myogenic cells within their ECM during those stages. Fibronectin is abundant in all interstitial tissues, while tenascin is restricted to intersegmental borders. Bundles of fibronectin and tenascin also penetrate into the myotome, possibly promoting myocyte alignment. A laminin matrix delineates the dermomyotome and myotome and undergoes dynamic changes, correlating with key developmental events. Conclusion: Our observations cast new light on how myotomal cells interact with their environment and suggest that, as the segmented myotomes transform into the epaxial muscle masses, the laminin matrix disassembles and myocytes use the abundant fibronectin matrix to reach their final organization. Developmental Dynamics 241:350–364, 2012. © 2011 Wiley Periodicals, Inc.     

The lack of genital ridge vascularization in the early chick embryo: Implications in the migration of the primordial germ cells

It is known that chick primordial germ cells (PGCs) in early embryonic development migrate via the blood vascular system to colonize the gonadal anlagen. Classically, two factors have been involved in the extravasation of PGCs from the blood stream: chemotactic and mechanical factors. An accurate knowledge of the vascular system of the genital ridge is therefore necessary. However, development of gonadal vascularization in bird embryos has been scarcely studied. Our previous studies have shown that the gonadal arteries develop from the mesonephric arteries. The purpose of this work was to study the implications of the development of the vascular system of the chick genital ridge on PGCs colonization. We selected the Hamburger and Hamilton (H-H) stage 18, since the genital ridge is well developed and PGCs actively extravasate. Forty chick embryos of this stage were processed for scanning electron microscopy of vascular corrosion casts and of critical point–dried specimens as well as light microscopy. Our results are conclusive. We could not find any vessel or capillary network supplying the genital ridge; the dorsal aorta and the primordia of the mesonephric arteries were the closest vessels. However, numerous interendothelial spaces were found in the dorsal aorta at the level of the genital ridge. It is suggested that the interendothelial gaps may be very important in the exchange of substances between the avascular genital ridge and the aortic endothelium at this developmental stage. Two different routes are thought to be involved in PGC migration to the gonadal anlage at this stage: the aortic endothelium and the mesonephric arteries. Whereas mechanical factors may be important for extravasation of PGCs in the mesonephric arteries, no reasons have been found from the morphological point of view to support a slowness of the blood flow in the dorsal aorta at the level of the genital ridge facilitating the extravasation. Anat. Rec. 251:398–405, 1998. © 1998 Wiley-Liss, Inc.     

The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro

The effect of iron and iron chelators on the development of the mouse embryo in vitro from the 1-cell stage to the blastocyst has been investigated. An adverse effect of iron was found. The high affinity iron chelator, desferal, also blocked development, whilst transferrin (whether as apoprotein or saturated with iron), DETAPAC and EDTA promoted development. The addition of transferrin permitted development to the blastocyst stage of embryos from stains normally exhibiting the 2-cell block. Under such circumstances both the rate of embryonic development and the proportion of embryos reaching the blastocyst stage approached levels found in vivo. Based on these results, a new medium, BAT6, is described for the optimal in-vitro culture of mouse embryos.     

Ioss of basement membranes in the invading front of O-1N, hamster squamous cell carcinoma with high potential of lymph node metastasis: An immunohistochemical study for laminin and type IV collagen

The change in immunohistochemical localization of the two basement membrane molecules, laminin and type IV collagen, was studied in relation to tumor growth and lymphatic invasion in the transplanted hamster squamous cell carcinoma, O-1N, that has a high potential of lymph node metastasis. At 1 week after transplantation, the tumors consisted of large round-shaped nests of approximately 200 mm in diameter, 80% of which were encircled by continuous laminin and type IV collagen positive lines representing the basement membrane. At 5 weeks, however, the tumor cell nests became half in diameter with invasion in small islands or cords at the periphery and only 40% of them had continuous basement membrane. At 10 weeks, the basement membranes were disrupted in varying degrees in approximately 90% of the tumor cell nests. The disruption was most conspicuous on the outer and invading side of the nests. Lymphatic invasion and lymph node metastasis were observed in animals after 5 weeks of transplantation and the immunoreactivity was absent around tumor cell clusters growing in lymphatic spaces. The findings indicate that the disappearance of basement membrane and reduction in size of tumor cell nests are essential signs for local invasion of tumor cells leading to lymphatic invasion and metastasis to regional lymph nodes.     

Morphogenesis of the node and notochord: The cellular basis for the establishment and maintenance of left–right asymmetry in the mouse

Establishment of left–right asymmetry in the mouse embryo depends on leftward laminar fluid flow in the node, which initiates a signaling cascade that is confined to the left side of the embryo. Leftward fluid flow depends on two cellular processes: motility of the cilia that generate the flow and morphogenesis of the node, the structure where the cilia reside. Here, we provide an overview of the current understanding and unresolved questions about the regulation of ciliary motility and node structure. Analysis of mouse mutants has shown that the motile cilia must have a specific structure and length, and that they must point posteriorly to generate the necessary leftward fluid flow. However, the precise structure of the motile cilia is not clear and the mechanisms that position cilia on node cells have not been defined. The mouse node is a teardrop‐shaped pit at the distal tip of the early embryo, but the morphogenetic events that create the mature node from cells derived from the primitive streak are only beginning to be characterized. Recent live imaging experiments support earlier scanning electron microscopy (SEM) studies and show that node assembly is a multi‐step process in which clusters of node precursors appear on the embryo surface as overlying endoderm cells are removed. We present additional SEM and confocal microscopy studies that help define the transition stages during node morphogenesis. After the initiation of left‐sided signaling, the notochordal plate, which is contiguous with the node, generates a barrier at the embryonic midline that restricts the cascade of gene expression to the left side of the embryo. The field is now poised to dissect the genetic and cellular mechanisms that create and organize the specialized cells of the node and midline that are essential for left–right asymmetry. Developmental Dynamics 237:3464–3476, 2008. © 2008 Wiley‐Liss, Inc.     

Comprehensive expression analysis for the core cell cycle regulators in the chicken embryo reveals novel tissue‐specific synexpression groups and similarities and differences with expression in mouse,frog and zebrafish

The core cell cycle machinery is conserved from yeast to humans, and hence it is assumed that all vertebrates share the same set of players. Yet during vertebrate evolution, the genome was duplicated twice, followed by a further genome duplication in teleost fish. Thereafter, distinct genes were retained in different vertebrate lineages; some individual gene duplications also occurred. To which extent these diversifying tendencies were compensated by retaining the same expression patterns across homologous genes is not known. This study for the first time undertook a comprehensive expression analysis for the core cell cycle regulators in the chicken, focusing in on early neurula and pharyngula stages of development, with the latter representing the vertebrate phylotypic stage. We also compared our data with published data for the mouse, Xenopus and zebrafish, the other established vertebrate models. Our work shows that, while many genes are expressed widely, some are upregulated or specifically expressed in defined tissues of the chicken embryo, forming novel synexpression groups with markers for distinct developmental pathways. Moreover, we found that in the neural tube and in the somite, mRNAs of some of the genes investigated accumulate in a specific subcellular localisation, pointing at a novel link between the site of mRNA translation, cell cycle control and interkinetic nuclear movements. Finally, we show that expression patterns of orthologous genes may differ in the four vertebrate models. Thus, for any study investigating cell proliferation, cell differentiation, tissue regeneration, stem cell behaviour and cancer/cancer therapy, it has to be carefully examined which of the observed effects are due to the specific model organism used, and which can be generalised.     

Analysis of gene expression in PTHrP−/− mammary buds supports a role for BMP signaling and MMP2 in the initiation of ductal morphogenesis

Parathyroid hormone–related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP's effects on mesenchymal cells, we profiled gene expression in WT and PTHrP^?/? mammary buds, and in WT and K14‐PTHrP ventral skin at E15.5. By cross‐referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds. Developmental Dynamics 238:2713–2724, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular mechanisms of lipopolysaccharide-induced cyclooxygenase-2 expression in human neutrophils: involvement of the mitogen-activated protein kinase pathway and regulation by anti-inflammatory cytokines

Neutrophils are an important cellular source of proinflammatory mediators, whose regulation may be of potential benefit for the treatment of a number of inflammatory diseases. However, the mechanisms of lipopolysaccharide (LPS)-induced neutrophil activation and its regulation by anti-inflammatory cytokines have not yet been fully elucidated. Recent studies have revealed that mitogen-activated protein kinases (MAPK) play a crucial role in the generation of proinflammatory mediators in some cell types. Therefore, we conducted this study to determine whether MAPK activation could be involved in prostaglandin E(2) (PGE(2)) production and cyclooxygenase (COX)-2 expression in LPS-stimulated human neutrophils. PD98059 (MEK1 inhibitor) and SB203580 (p38(MAPK) inhibitor) reduced PGE(2) production as well as COX-2 expression in LPS-stimulated neutrophils. In addition, both extracellular signal-regulated protein kinase (ERK) and p38(MAPK) were phosphorylated and activated in time- and dose-dependent manners. Since we previously showed that IL-10 and IL-4 similarly inhibited COX-2 expression in LPS-stimulated neutrophils, we next tested the effects of IL-10 and IL-4 on the phosphorylation and activation of both kinases. IL-10 inhibited the phosphorylation and activation of p38(MAPK), but not ERK. In addition, IL-4 caused a marginal inhibition in the activation of p38(MAPK). Taken together, these results suggest that both ERK and p38(MAPK) pathways are involved in LPS-induced COX-2 expression and PGE(2) production in neutrophils, and IL-10 and IL-4 inhibit neutrophil prostanoid synthesis by down-regulating the activation of p38(MAPK).     

Molecular concepts in the pathogenesis of ameloblastoma: Implications for therapeutics

Ameloblastoma is a benign odontogenic neoplasm that may exhibit aggressive biological behavior as evidenced by its rapid growth and significance recurrence rates following initial surgical resection. Currently, the only therapy for ameloblastoma is surgical, and adjunctive treatment modalities are needed to mitigate tumor growth and to reduce the need for extensive and disfiguring surgeries. Many studies have identified markers expressed by ameloblastoma and these lend insight to our understanding of tumor progression. This review provides a summary of the specific molecular pathways implicated in tumor pathogenesis, including those involved in bone remodeling, apoptosis, cell signaling, and tumor suppression. Based on these data, we identify several prognostic or therapeutic markers that have been used successfully in the treatment of other neoplastic processes that may also have diagnostic and prognostic utility for ameloblastoma. Thus, it is important to determine which markers hold the greatest promise for clinical management of this benign neoplasm in order to improve treatment options, particularly in patients with aggressive forms of ameloblastoma.     

Studies for a genotoxic potential of some endogenous and exogenous sex steroids. II. communication: Examination for the induction of cytogenetic damage using the chromosomal aberration assay on human lymphocytes in vitro and the mouse bone marrow micronucleus test in vivo

The cytogenetic potential of 10 sex steroids (cyproterone acetate, drospirenone, gestodene, cyclodiol, cyclotriol, ethinylestradiol, atamestane, lilopristone, onapristone and propylmesterolone) with various medical indications was determined using the chromosomal aberration test in human lymphocytes in vitro and the mouse bone marrow micronucleus test in vivo. Nine of these sex steroids (gestodene was omitted) were investigated in the human lymphocyte assay and found to be negative with respect to the induction of chromosomal aberrations either with or without metabolic activation. In all assays the highest concentration evaluated was either clearly cytotoxic or, in case of noncytotoxicity, resulted in visible precipitates in the culture medium. Evaluation of the data from the mouse bone marrow micronucleus test indicated that the seven steroids (cyproterone acetate, drospirenone, gestodene, ethinylestradiol, atamestane, onapristone and propylmesterolone) investigated failed to induce enhanced frequencies of micronucleated polychromatic erythrocytes in male and female mice. The steroids were tested up to dose levels which induced signs of toxicity in the experimental animals or, in the case of non toxic compounds, the animals were treated up to the maximum recommended dose of 2 g/kg body weight. Evaluation of all data indicates that the investigated estrogens, progestins and other sex steroids had no genotoxic potential detectable with the chromosomal aberration assay on cultured human lymphocytes or the mouse bone marrow micronucleus test. © 1996 Wiley-Liss, Inc.     

An emerging role of neutrophils and NETosis in chronic inflammation and fibrosis in systemic lupus erythematosus (SLE) and ANCA-associated vasculitides (AAV): Implications for the pathogenesis and treatment

Neutrophils derive from hematopoietic stem cells (HSCs) with systemic inflammation driving their activation and differentiation to myeloid progenitors to ensure enhanced myelopoiesis. Epigenetic reprograming and re-education of these HSCs produces neutrophils primed towards elimination of pathogens and increased inflammatory response. Neutrophils -an important component of acute inflammation- are not present in chronic inflammatory tissues leading to the false assumption that they may not be as important for the latter. Activated neutrophils may release Neutrophil Extracellular Traps (NETs) during a distinct form of cell death, named NETosis; NETs are rich in bioactive molecules that promote thrombosis (including atherothrombosis), inflammation and fibrosis. Thus, although neutrophils may not be present in chronic inflammatory lesions, their remnants may amplify the inflammatory response beyond their short life-span in the tissues. Herein, we review current evidence supporting a role of neutrophils and NETosis in tissue injury and dysfunction in systemic autoimmunity using as disease paradigms Systemic Lupus Erythematosus (SLE) and the ANCA-associated vasculitides (AAV). We also discuss the mechanisms involved and their potential as targets for novel therapy and drug repositioning.     

Inflammatory level under different p53 mutation status and the regulation role of curcumin in tumor microenvironment

The inflammation is tightly associated with tumor development, promoting or inhibiting tumorigenesis. And mutant p53 is speculated to promote inflammation and tumorigenesis. The tumor associated macrophages are usually educated to present the anti-inflammatory profile to tune down antitumor immunity. However, the impact of p53 mutants on macrophages is not clear. Here, we compared the basal inflammatory level and macrophage profiles in tumor cells and tumor samples with different p53 mutations. Data revealed that a lower inflammatory level was maintained in immune organs and tumor cells with p53 point mutations than those with p53 null mutation. Using the tumor cell-derived conditional media to culture macrophages, we found that the media from cells with p53 mutations, especially the point mutations, could decrease M1 markers and inhibit phagocytosis, suggesting the p53 mutation promoted M2 profile polarization.To target the p53 mutation induced M2 macrophage polarization, we applied low-concentration curcumin to the tumor cells with different p53 mutations. The data showed that curcumin could inhibit STAT3 signal and decrease PPARγ and CSF1 in tumor cells and tumor samples. In vitro, the co-culture assays showed that the curcumin treatment shifted p53 mutation educated macrophages back towards M1 profile. In vivo, the curcumin-treated MEFs showed obvious tumor inhibition, and the tumor samples displayed inhibited M2 markers. Results suggested that curcumin could inhibit p53 mutation educated macrophage induction and suppress M2-promoted tumorigenesis.Our study illustrated the inflammatory level under different p53 status and the inflammatory regulated role of curcumin in tumor environment. This study might provide a potential method in tumor personalized treatment aiming immune therapy in different p53 status.