Immunolocalization of a nuclear protein bound to the sphere organelle during oogenesis and embryogenesis inPleurodeles waltl

Summary The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.     

Transient coexpression of desmin and cytokeratins 8 and 18 in developing myocardial cells of some vertebrate species

During myogenesis the intermediate-sized filament (IF) cytoskeleton is characterized by increasing proportions of desmin. While skeletal and smooth muscle formation occurs in free mesenchymal cells containing vimentin-type IFs, myocardial development starts from a polar epithelium containing cytokeratin IFs and desmosomes. Therefore, we have studied the formation of the epicardium and the myocardium in different vertebrate species, combining light and electron microscopic immunolocalization techniques with gel-electrophoretic analyses of cytoskeletal proteins of microdissected myocardial tissue at differing developmental stages. In this report, we describe results obtained from advanced stages of myocardial differentiation. In all species studied the myocardial cell possess IFs abundant in desmin, often together with smaller amounts of vimentin, and the mesothelial layer of the epicardium contains cytokeratin IFs. However, we have observed remarkable interspecies differences with respect to the occurrence of cytokeratins in embryonic myocardial cells. In fetal human myocardium, from week 10 of pregnancy on, but not in juvenile and adult myocardium, and in chicken myocardium of all stages examined (until several days after hatching) specific immunostaining was seen with certain broad-range cytokeratin antibodies as well as with antibodies specific for cytokeratins 18 (in both species) and 8 (showing significant reaction only in human). This cytokeratin immunoreaction, however, did not appear in IFs extending throughout the cytoplasm or at Z-lines, but was localized in punctate arrays representing aggregates of dense material. The aggregates were often enriched at, but not restricted to, the desmosomal plaques of the intercalated discs. These observations were supported by gel-electrophoretic demonstration of small but significant amounts of cytokeratins 18 (in both species) and 8 (detected only in human) in microdissected myocardial tissue. We also observed cytokeratins in smooth muscle cells of some cardiac blood vessels. In contrast, bovine myocardium of advanced fetal age as well as rat and mouse myocardium (from fetal day 12 on) were negative for cytokeratins with all methods, although epicardial cytokeratin IFs were demonstrable. These observations are discussed in relation to myocardial histogenesis and to general problems of cytokeratin gene expression control in epithelial and nonepithelial cells.     

Morphology of developing heart in cardiac lethal mutant Mexican axolotls, Ambystoma mexicanum

In Ambystoma mexicanum, recessive mutant gene c results in an absence of embryonic heart function because of altered influences from surrounding tissues (Humphrey, 1972). The present light and electron microscope study compares heart development in normal and mutant embryos from Harrison stage 34 or 6 days (at which normal heart beat initiates) through stage 41 or 25 days (at which mutant embryos die). The hearts display increasing differences as development progresses, and by stage 41 mutant abnormalities are striking. The normal myocardium shows organized sarcomeres at stage 34 and numerous intercalated discs subsequently appear. By stage 41, the normal myocardium is composed of highly differentiated muscle cells and shows extensive trabeculation. The mutant myocardium throughout development remains only one cell layer thick with no indication of developing trabeculae. Mutant cells at stage 34 have a few 140 Å and 60 Å filaments along with what appear to be Z bodies. A partial organization of myofibrillar components is occasionally noted at stages 38–41; however, distinct sarcomeres are not apparent and intercalated discs are rarely seen. In general the mutant cells appear less differentiated than usual and in many respects are reminiscent of pre-heart-beat normal cells. Although most mutant cells show images characteristic of pathological conditions (e.g., pleomorphic mitochondria, membranous whorls, and numerous autophagic vacuoles), selective myocardial cell death, a phenomenon associated with normal trabeculation, is not evident. It is clear that gene c, in homozygous condition, results in an altered pattern of heart cell differentiation. The mutation, by way of abnormal inductive processes, appears to affect the synthesis and organization of heart contractile proteins.     

Immunofluorescent studies on titin and myosin in developing hearts of normal and cardiac mutant axolotls

Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34–35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26–35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35–42. At tailbud stage-26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32–33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages. © 1994 Wiley-Liss, Inc.     

FGF10/FGFR2b signaling is essential for cardiac fibroblast development and growth of the myocardium

The epicardium serves as a source of growth factors that regulate myocardial proliferation and as a source of epicardial-derived cells (EPDC), which give rise to interstitial cardiac fibroblasts and perivascular cells. These progenitors populate the compact myocardium to become part of the mature coronary vasculature and fibrous skeleton of the heart. Little is known about the mechanisms that regulate EPDC migration into the myocardium or the functions carried out by these cells once they enter the myocardium. However, it has been proposed that cardiac fibroblasts are important for growth of the heart during late gestation and are a source of homeostatic factors in the adult. Here, we identify a myocardial to epicardial fibroblast growth factor (FGF) signal, mediated by FGF10 and FGFR2b, that is essential for movement of cardiac fibroblasts into the compact myocardium. Inactivation of this signaling pathway results in fewer epicardial derived cells within the compact myocardium, decreased myocardial proliferation and a resulting smaller thin-walled heart.     

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.     

H-Type 1 carbohydrate antigen expression by ovine endometrial cells

Our objective was to determine whether H-Type 1 carbohydrate antigen is expressed by ovine endometrial epithelial cells. Endometrium was obtained from sheep on days (D) 1, 5, 11, 13, and 15 of the estrous cycle or pregnancy and D17 and D19 of pregnancy. Immunofluorescence microscopy of frozen tissue sections revealed intense staining on the apical surface of glandular uterine epithelial (GE) cells from D11 to D17 of pregnancy. Light punctate staining of luminal uterine epithelial (LE) cells was present from D15 to D19 of pregnancy, with isolated areas of intense staining observed only on D15 of pregnancy. There were no noticeable differences in staining patterns on equivalent d of the estrous cycle. Immortalized sheep LE and GE cells were used to determine whether estradiol (E), progesterone (P), or E + P, with or without interferon tau (IFNtau), regulates H-Type 1 antigen expression in vitro. Intermittent punctate surface staining was observed on both cell lines independent of steroid treatment. Treatment with P or IFNtau increased H-Type 1 antigen expression (P < 0.01) and resulted in large aggregates of punctate staining. Domain-specific biotinylation and Western blotting of cell lysates from LE and GE cells were used to identify proteins carrying the H-Type 1 antigen. For both cell types, major immunoreactive apical membrane proteins were detected at 31, 33, 42, 55, 60, and 70 kDa. Therefore, the H-type 1 antigen is expressed mainly on GE cells during pregnancy recognition in utero and up-regulated by P and IFNtau on LE and GE cells in vitro.     

调节性T细胞参与调控新生小鼠心肌再生

为探究调节性T（regulatory T,Treg）细胞在新生小鼠心肌损伤后再生中的作用,首先建立新生小鼠心肌再生模型。C57BL/6J（C57）新生1 d小鼠20只随机分成2组。实验组进行心尖切除（apex resection,AR）,假手术（Sham,SH）组只进行开胸。术后7 d取心脏组织,利用在细胞核表达的增殖标志物磷酸化组蛋白H3（phospho-histone H3,pH3）和Ki67分别与在心肌细胞胞质特异表达的α-辅肌动蛋白（alpha-actinin cytoskeletal isoform,α-actinin）,进行免疫共染检测心肌细胞增殖。结果显示,与SH组相比,AR组pH3+及Ki67+的心肌细胞明显增多。而且Masson三色染色结果显示,术后21 d被切除的心肌组织完全再生。为研究Treg细胞是否参与调控新生小鼠心肌损伤后的再生,Western印迹检测Treg细胞特异转录因子叉头/翼状螺旋转录因子3（forkhead box P3,Foxp3）蛋白表达水平。结果显示,术后7 d、14 d,AR组心和脾中Foxp3与SH组相比显著升高（P<0.05）。同时,免疫组化染Foxp3结果显示,术后7 d、14 d, AR组与SH组相比,心尖处有大量的Treg细胞富集。为更直观地检测AR后Treg细胞的数目变化,利用流式细胞仪检测术后7 d Treg细胞数目。结果显示,AR组心和脾中Treg细胞数目与SH组相比显著增多（P<0.01）。为研究Treg细胞对AR后心肌再生的影响,引入注射白喉毒素（diphtheria toxin,DT）的Foxp3DTR小鼠,可特异性敲除Treg细胞。实时定量PCR结果显示,AR+DT组与AR+PBS组相比,抑炎因子白介素IL（interleukin,IL）-10、IL-13与转化生长因子TGF（transforming growth factor,TGF）-β表达均降低（P<0.05,P<0.01,P<0.01）。而促炎因子IL-6、IL-1β和肿瘤坏死因子-α（tumor necrosis factor,TNF-α）表达均升高（P<0.01,P<0.001,P<0.01）。免疫荧光染色检测结果显示,AR+DT组与AR+PBS组相比,术后7 d pH3+及Ki67+的心肌细胞明显减少;并且Masson三色染色结果显示,术后21 d AR+DT组被切除的心肌组织不能再生。综上所述,敲除Treg细胞会加剧AR后的炎症反应,抑制心肌细胞增殖,最终导致新生小鼠心肌再生能力丢失。     

Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice

Transformation of endocardial endothelial cells into invasive mesenchyme is a critical antecedent of cardiac cushion tissue formation. The message for bone morphogenetic protein (BMP)-2 is known to be expressed in myocardial cells in a manner consistent with the segmental pattern of cushion formation [Development 109(1990) 833]. In the present work, we localized BMP-2 protein in atrioventricular (AV) myocardium in mice at embryonic day (ED) 8.5 (12 somite stage) before the onset of AV mesenchymal cell formation at ED 9.5. BMP-2 protein expression was absent from ventricular myocardium throughout the stages examined. After cellularization of the AV cushion at ED 10.5, myocardial BMP-2 protein expression was diminished in AV myocardium, whereas cushion mesenchymal cells started expressing BMP protein. Expression of BMP-2 in cushion mesenchyme persisted during later stages of development, ED 13.5-16, during valuvulogenesis. Intense expression of BMP-2 persisted in the valve tissue in adult mice. Based on the expression pattern, we performed a series of experiments to test the hypothesis that BMP-2 mediates myocardial regulation of cardiac cushion tissue formation in mice. When BMP-2 protein was added to the 16-18 somite stage (ED 9.25) AV endocardial endothelium in culture, cushion mesenchymal cells were formed in the absence of AV myocardium, which invaded into collagen gels and expressed the mesenchymal marker, smooth muscle (SM) alpha-actin; whereas the endothelial marker, PECAM-1, was lost from the invaded cells. In contrast, when noggin, a specific antagonist to BMPs, was applied together with BMP-2 to the culture medium, AV endothelial cells remained as an epithelial monolayer with little expression of SM alpha-actin, and expression of PECAM-1 was retained in the endocardial cells. When noggin was added to AV endothelial cells cocultured with associated myocardium, it blocked endothelial transformation to mesenchyme. AV endothelium treated with BMP-2 expressed elevated levels of TGFbeta-2 in the absence of myocardium, as observed in the endothelium cocultured with myocardium. BMP-2-supported elevation of TGFbeta-2 expression in endocardial cells was abolished by noggin treatment. These data indicated that BMP signaling is required in and BMP-2 is sufficient for myocardial segmental regulation of AV endocardial cushion mesenchymal cell formation in mice.     

Initial expression of type I procollagen in chick cardiac mesenchyme is dependent upon myocardial stimulation

Formation of the atrioventricular (AV) mesenchyme is a critical step in early heart development. Endothelial cells are activated and transformed into a mesenchymal population that invades the cell-free myocardial basement membrane. This process can be duplicated in collagen gel culture, where it has been established that myocardium or its secretory products activate the endothelium. The purpose of the present study was to determine when these activated endothelial and/or mesenchymal cells start producing type I collagen in situ. These results were compared to those obtained from a culture model of mesenchyme formation. The production of type I collagen was monitored using a monoclonal antibody (M38) that recognizes the carboxy-terminal propeptide of human type I procollagen. The initial expression of the latter within activated AV endothelial and mesenchymal cells in ovo was 48 hr following activation. Prior to this time, only the myocardium was reactive with M38. AV explants of early hearts on collagen gels revealed staining of activated endothelial and mesenchymal cells with M38 after 48 hr in coculture with myocardial tissue. Explants that were prevented from activating (myocardium removed) never expressed the M38 antigen. Similarly, AV endothelial monolayers grown in the presence of myocardial conditioned medium activated and expressed type I collagen after 48 hr in culture, whereas those grown in standard medium did not. These results establish the initial expression of type I collagen within activated AV endothelium and mesenchyme. In addition, the data suggest that the expression of type I collagen within the AV mesenchyme may be dependent on extrinsic influences that induce the AV endothelium to transform into mesenchyme.     

Temporal and spatial characteristics of apoptosis in the infarcted rat heart

Following myocardial infarction (MI), tissue repair/remodeling occurs in both the infarcted and noninfarcted myocardium. Apoptosis has been demonstrated to play an important role in these processes. In the present study, we sought to determine the temporal and spatial characteristics of apoptosis in the infarcted heart as well as to identify cells undergoing programmed cell death at different stages of repair/remodeling and their relationship to the expression of anti-/pro-apoptotic genes following MI. Our study has shown that apoptosis appears in both infarcted and noninfarcted myocardium, and cells undergoing apoptosis depend on the stage of healing. In the infarcted myocardium, apoptosis contributes to the loss of cardiomyocytes during the early stage of healing, elimination of inflammatory cells during the inflammatory phase of healing, and reduction of myofibroblasts with the fibrogenic phase of repair in the infarcted myocardium. In noninfarcted myocardium, cardiomyocyte apoptosis was observed from day 3 to 28 postMI. Cardiac apoptosis following MI is correlated with the increase of Bax expression.     

炎症因子、生长因子以及凋亡因子在压疮慢性难愈合性创面中的表达及作用

目的：探讨炎症反应、生长因子及凋亡因子在压疮慢性创面中的表达及作用。方法：选取2013年10月至2015年7月河南大学第一附属医院收治的患者,其中临床Ⅲ、Ⅳ期压疮患者共20例,急性创面10例,正常皮肤组织6例。通过HE染色观察不同创面组织的形态学特征;免疫组织化学法检测组织中细胞凋亡因子Caspase-3的分布规律;荧光定量PCR法定量分析IL-1β、IL-6、TNF-α、VEGF、bFGF及其受体KDR、FGFR1基因水平的变化特征。结果：Ⅲ、Ⅳ期压疮创面中可见炎性细胞浸润;凋亡信号因子caspase-3在压疮组中的表达高于其他两组,差异有统计学意义;IL-1β、IL-6、TNF-α表达高于急性创面组和正常皮肤组;VEGF和bFGF生长因子及其受体KDR和bFGFR1表达分别低于对照组。结论：炎症因子和凋亡因子在压疮慢性创面中持续长时间的高表达、生长因子及其受体显著的低表达可能是压疮慢性难愈合性创面形成的机制和难以彻底治愈的重要因素。     

Serological characterization of a nervous system/sperm antigen (NS-52): demonstration of its presence on murine preimplantation embryos.

Anti-NS-5 antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice could be shown to recognize two cell surface antigens on cerebellar cells, NS-5₁ and NS-5₂, the latter antigen being shared with mouse and rat but not rabbit sperm. An antigen operationally identical to NS-5₂ was detected using indirect immunofluorescence staining on mouse preimplantation stages of development. While the unfertilized ova did not express detectable antigen on the cell surface, the fertilized egg expressed antigen shortly before the first cleavage division. From that stage onward, the anti-NS-5 antiserum stained the blastomeres of all stages, including the trophoblast cells and inner cell mass cells of the blastocyst. No difference in staining activity was observed for preimplantation embryos of various mouse strains analyzed: C57BL/6J, BALB/c, 129/J, C3H/DiSn, CKB × BALB.K, C3H.SW/Sn, and Swiss Webster mice. The staining activity was removed when the antiserum was preabsorbed with cerebellum or sperm from any of these mouse strains or with cerebellum and sperm of rats. Lymphocytes, thymocytes, liver, kidney, and skeletal muscle from early postnatal and adult mice and heart from early postnatal mice did not absorb the staining activity and neither did rabbit sperm nor cerebellum.     

An appropriate loading control for western blot analysis in animal models of myocardial ischemic infarction

An appropriate loading control is critical for Western blot analysis. Housekeeping proteins (HKPs), such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin, are commonly used to normalize protein expression. But HKP expression can be impacted by certain experimental conditions, such as ischemic myocardial infarction. This study was undertaken to look for an appropriate loading control for western blot analysis of ischemic myocardium. Myocardial ischemic infarction was induced by left anterior descending coronary artery (LAD) ligation in Rhesus monkeys and C57BL/6 mice. The heart tissue samples from different areas and time points after surgery were subjected to western blot or gel staining. The level of β-actin, GAPDH, β-tubulin, and total protein were tested. The total protein level was consistent in all groups, whereas the protein level of β-tubulin and β-actin were different in all groups. However, the protein level of GAPDH was stable in the Rhesus monkey model. We concluded that total protein was the most appropriate internal control in different stages of myocardial ischemic disease of various animal models. GAPDH is a reliable internal control only for ischemic myocardium of Rhesus monkey.     

The human homologue of the Aspergillus nuclear migration gene nudC is preferentially expressed in dividing cells and ciliated epithelia

An early event in apoptosis is exposure of phosphatidylserine, an aminophospholipid normally present in the inner leaflet of the plasma membranes, at the outer leaflet of the plasma membrane facing the extracellular space. Annexin V (Anx-V) is a 35-kDa protein with high affinity for phosphatidylserine, which can be applied to detect apoptosis. We injected biotin-labelled Anx-V intravenously in adult mice and examined the tissue distribution of Anx-V-labelled cells in dental and periodontal tissues using ABC-peroxidase histochemistry. In the continuously erupting incisors, strong and frequent immunostaining was observed in transitional stage and late maturation stage ameloblasts with less frequent staining in preameloblasts. Frequency of staining in odontoblasts and pulp cells was low but increased slightly at older stages of dentinogenesis. Labelling was also seen in phagocytic or phagocytic-like cells in the enamel organ and pulp. A positive staining was furthermore found in fibroblasts of the periodontal ligament in continuously erupting incisors and in fully erupted molar teeth. Staining intensity and the number of positive cells were enhanced by antigen retrieval using high-pressure cooking. We conclude that Anx-V-biotin labels dental cells in early stages of cell death and indirectly cells that have ingested labelled apoptotic cells during the course of the experiment. The data confirm that during amelogenesis most cell death occurs in transitional stage and late maturation stage ameloblasts. Thus, labelling with Anx-V is a useful marker for studying cell death and the dynamics of clearance of apoptotic cells during tooth development.     

Role of myocardial hypoxia in the remodeling of the embryonic avian cardiac outflow tract

The embryonic cardiac outflow myocardium originates from a secondary heart-forming field to connect the developing ventricles with the aortic sac. The outflow tract (OFT) subsequently undergoes complex remodeling in the transition of the embryo to a dual circulation. In avians, elimination of OFT cardiomyocytes by apoptosis (stages 25-32) precedes coronary vasculogenesis and is necessary for the shortening of the OFT and the posterior rotation of the aorta. We hypothesized that regional myocardial hypoxia triggers OFT remodeling. We used immunohistochemical detection of the nitroimidazole EF5, administered by intravascular infusion in ovo, as an indicator of relative tissue oxygen concentrations. EF5 binding was increased in the OFT myocardium relative to other myocardium during these stages (25-32) of OFT remodeling. The intensity of EF5 binding paralleled the prevalence of apoptosis in the OFT myocardium, which are first detected at stage 25, maximal at stage 30, and diminished by stage 32. Evidence of coincident hypoxia-dependent responses included the expression of the vascular endothelial growth factor (VEGF) receptor 2 by the OFT myocardium, the predominant expression of VEGF122 (diffusible) isoform in the OFT, and the recruitment of QH1-positive pro-endothelial cells to the OFT and vasculogenesis. Exposure of embryos to hyperoxia (95% O(2)/5% CO(2)) during this developmental window reduced the prevalence of cardiomyocyte apoptosis and attenuated the shortening and rotation of the OFT, resulting in double-outlet right ventricle morphology, similar to that observed when apoptosis is directly inhibited. These results suggest that regional myocardial hypoxia triggers cardiomyocyte apoptosis and remodeling of the OFT in the transition to a dual circulation, and that VEGF autocrine/paracrine signaling may regulate these processes.     

Hepatopancreas is the likely organ of vitellogenin synthesis in the freshwater prawn, Macrobrachium rosenbergii

The objective of the present study was to investigate the source of vitellogenin in the freshwater prawn, Macrobrachium rosenbergii. Ovarian development of M. rosenbergii was classified into five stages (stage I-V). Vitellin/vitellogenin was detected in the ovary and the hepatopancreas in different stages by native-PAGE and Western blotting. Two and three subunits of vitellin were observed in the ovary at the early- (I-II), mid- and late- (III-V) stages, respectively. The subunit of vitellogenin was not detected in the hepatopancreas at different stages of prawns. Hepatopancreas had positive immunocytological staining (against vitellin antibody) in different ovarian stages of prawn. Only vitellogenic oocyte but not previtellogenic oocytes and follicle cells had a positive immunocytological staining. Hepatopancreas could synthesize radiolabeled immunoreactive proteins after incubation with radiolabeled glycine on the basis of immunoprecipitation (against vitellin antiserum). Therefore, it is concluded that hepatopancreas is the most likely organ to synthesize vitellogenin in the freshwater prawn, M. rosenbergii.     

Expression of vinexin alpha in the dorsal half of the eye and in the cardiac outflow tract and atrioventricular canal

The Smads are intracellular signalling molecules that transduce signals from receptors for members of the TGF-beta superfamily to the nucleus. We have cloned the Xenopus orthologue of Smad3 (XSmad3). It is 94.6% identical to human Smad3 at the amino acid level. It is expressed as a maternal mRNA which disappears after stage 10.5, but reappears at the early tailbud stages. It is much less abundant than XSmad2 at the early developmental stages. From Stage 27 onwards XSmad3 is expressed with XSmad2 throughout the head region and in the somitic region. Strikingly however, XSmad3 alone is specifically expressed in the chordoneural hinge, the notochord and in the developing heart. Closer analysis reveals that XSmad3 is specifically expressed in the endocardium but not in the myocardium or pericardium. The chordoneural hinge staining persists at least until stage 40 whereas the staining in the endocardium peaks at approximately stage 32/33.