Early chick limb cartilaginous elements possess polarizing activity and express hedgehog-related morphogenetic factors

Skeletal patterning and morphogenesis in the developing limb are thought to be regulated by instructive factors and cues from the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the dorsal ectoderm. However, the activities of the ZPA and AER dwindle early in embryogenesis and soon after ceases, when in fact the proximal skeletal elements are still rudimentary in structure and the more distal ones are yet to become recognizable. Thus, we asked whether the chondrocytes emerging within each mesenchymal condensation may themselves start expressing properties similar to those of ZPA and/or AER and, in so doing, may bring skeletal development to completion. Indeed, we found that the cartilaginous, but not precartilaginous, tissues in early chick limbs possess ZPA-like properties. They expressed an endogenous factor related to Sonic hedgehog (Shh), most likely Indian hedgehog (Ihh), and when fragments were grafted to the anterior margin of host stage 16-20 chick wing buds, they induced supernumerary skeletal elements (polarizing activity). The acquisition of polarizing activity by the cartilaginous structures followed clear proximo-to-distal and posterior-to-anterior routes. Thus, (1) stage 25 cartilaginous humerus had polarizing activity while stage 25 prospective radius did not, (2) posteriorly-located stage 29 ulna had stronger activity than anteriorly-located stage 29 radius, and (3) ulna's diaphysis had stronger activity at stage 29 than 31 while radius's diaphysis was stronger at stage 31 than 29. Prior to inducing extra digit formation, the cartilaginous grafts induced Hoxd-12 and Hoxd-13 gene expression in adjacent competent mesenchymal tissue. Strikingly, the cartilaginous grafts activity also expression of Shh and polarizing activity in adjacent mesenchyme, which ZPA grafts cannot do; thus, the cartilaginous structures displayed activities "upstream" of those of the ZPA. The results support our hypothesis that chondrocytes may themselves direct skeletal morphogenesis. In so doing and as a result of their inductive activities, the cells may also have an important role in the completion of limb patterning and morphogenesis.     

The development of cartilaginous wrist in human fetuses

Wrist bones were studied in fetuses aged 9 to 24 weeks. In fetuses aged 9 weeks the primordia of all wrist bones are present. The joint cavities begins to form early in the 3rd month.     

Laminin chain expression by chick chondrocytes and mouse cartilaginous tissues in vivo and in vitro

We have observed that laminins are expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and adult mouse knee joint by the methods of in situ hybridization, immunohistochemistry, Western blotting, and immunoprecipitation. From in situ hybridization using similar sized RNA probes for different mouse laminin chains, mRNAs for the alpha 1, alpha 2, beta 1, beta 2, and gamma 1 chains were expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and the articular cartilage cap and epiphyseal growth plate of adult mouse knee joint. Through the use of chain-specific antibodies, staining for laminins was observed in the cytoplasm of chondrocytes from chick embryo sternum, mouse limb bud, and adult mouse knee joint. Western blot analysis confirmed the presence of laminin chains in the cells and sternal tissues. Cultured chick embryonic sternal chondrocytes expressed laminin mRNAs in the proliferating stage (2-3 days of culture) but the level increased in the aggregated cells during the maturation stage (5-7 days of culture). Comparable data were also obtained after immunostaining the cells. Thus, laminins are expressed in significant amounts by chondrocytes and may have an important role in cartilage development.     

Neurotrophin-3 antibodies disrupt the normal development of the chick retina

When chick embryos are treated with a monoclonal antibody specifically blocking the activity of neurotrophin-3 (NT-3), the development of the retina is profoundly affected. Fewer axons are found in the optic nerve, and the retina shows abnormalities in all layers. Early during retinogenesis, the proportion of dividing cells is higher in NT-3-deprived embryos compared with age-matched controls and that of differentiated neurons is smaller. The NT-3 receptor trkC is expressed early by a majority of retinal cells, and NT-3 is present in the retina at the earliest stage studied. Initially, it is located mainly in the pigmented epithelium, with a shift toward the neural retina as development proceeds. Thus, NT-3 seems to be an essential intrinsic signal acting early in development to promote the differentiation and survival of many retinal neurons.     

Contactin/F11 and tenascin-C co-expression in the chick retina correlates with formation of the synaptic plexiform layers

The neural immunoglobulin-like cell adhesion molecule contactin/F11 and the extracellular matrix glycoprotein tenascin-C are prominent molecules in the developing nervous system which interact in in vitro assays (Zisch et al., J. Cell Biol. 119, 203-213). To determine their potential role in neural development, the distribution of tenascin-C and contactin/F11 was examined in the developing chick retina. The onset of both tenascin-C and contactin/F11 expression coincides with the appearance of ganglion cell dendrides and neurites from bipolar and amacrine cells in the inner layer (IPL) at E8, and the extension of bipolar and horizontal cell processes in the outer plexiform layer (OPL) at E9. Contactin/F11 expression is co-ordinately upregulated with the TN190 and TN200 tenascin-C isoforms between embryonic day 8 (E8) and E17, while little, if any, of the TN220 isoform, which does not bind contactin/F11, is detected. In situ hybridization reveals that tenascin-C and contactin/F11 mRNAs are synthesized by different neuronal types. Tenascin-C mRNA probes hybridize to amacrine and displaced amacrine neurons, and horizontal neurons. In cultured retinal cells, tenascin-C is also present on process-bearing neurofilament-positive cells. Contactin/F11 mRNA is detected in bipolar cells or their precursors from E8-9, and later in horizontal and ganglion neurons. The highest levels and greatest overlap in the synaptic IPL and OPL are reached at E17, when the stratification of the retina is nearly complete. These results are consistent with a putative role for contactin/F11-tenascin-C interactions in the establishment of synaptic layers in the retina.     

Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs

BACKGROUND: High exposure to house dust mite allergen during the first year of life has been found to increase the risk of subsequent asthma and mite sensitization. Environmental factors, home construction and cleaning methods used are associated with levels of dust mites in the home. OBJECTIVE: To investigate determinants of levels of Der p 1 and Der f 1 mite allergens in homes of infants in southern Tasmania. METHODS: Dust samples were collected from 72 homes of infants participating in the Tasmanian Infant Health Survey (TIHS). The Der p 1 and Der f 1 allergen concentrations in these samples were measured. The TIHS interviewers obtained information from the mothers of the infants via a questionnaire, observed specified aspects of the home environment, and took readings of bedroom temperature and humidity. The effect of each item on allergen concentration in dust from bedroom floors was examined in a variety of ways. Those items which in this study appeared to be significantly related to allergen concentrations plus items which in other studies have been found to be related to allergen concentrations were then investigated further in multivariate models. RESULTS: Der p 1 allergen concentration (microg/g) and density (microg/m2) in dust from bedroom floors were found to be related to several home environment factors. In the univariate analyses, indoor humidity, 24 h maximum temperature, number of residents and a combination of floor covering and cleaning methods appeared to have a significant effect on allergen levels. These factors remained important in the multivariate model except that indicators for mould in the bathroom and drying washing on an outside line replaced indoor humidity. CONCLUSION: Features related to home dampness, the number of residents and floor covering and cleaning were major determinants of Der p 1 levels in the bedrooms studied.     

Studies on growth, development, and infectivity of Philophthalmus hegeneri Penner and Fried 1963 in the chick

A reinvestigation of the growth curve of Philophthalmus hegeneri in chicks showed rapid growth for the first 17 days after infection. After this, the growth rate declined for multiple infections (two or more worms per eye), and after 20 days, adults in monometacercarial infections ceased appreciable growth and never attained reproductive maturity. These results are similar to those reported by Fried (1962), but here adults were approximately 0.5 mm shorter in all cases and were slower in producing mature eggs. When worms from 28 to 36 days old from monometacercarial infections were transplanted into multiple infections, normal growth occurred. Worms recovered from multiple infections and transplanted singly grew normally for at least 18 days but recovery rates were extremely low. When P. hegeneri adults over 20 days old from monometacercarial infections were transplanted singly with Philophthalmus megalurus adults, growth was not stimulated in either species.     

Retinoic acid-induced abnormal development of hindlimb joints in the mouse

We asked if the amount of SP-B (range 37-410 micrograms/ml) in surfactants used to treat preterm lambs at 123 days of gestation correlated with postnatal lung function or the SP-B content of surfactant recovered by alveolar washes after 10 h ventilation. Ventilation was initiated using a low tidal volume strategy to minimize early lung injury. There were small increases in compliance for the lambs treated with surfactants containing more than 37 micrograms/ml SP-B and an increased lung volume for lambs treated with a surfactant containing 385 micrograms/ml/ml relative to the surfactant containing 37 micrograms/ml SP-B. The amount of SP-B in the surfactant used for treatment correlated linearly with the amount of SP-B in the surfactant recovered from the lambs (r = 0.95, p < 0.001). Albumin leak from the vasculature to the airspace was low as was total protein in alveolar washes, indicating minimal lung injury. The SP-B content of surfactant (from 37 to 410 micrograms/ml) had minimal effects on postnatal lung function over a 10-hour study period in lambs ventilated in a manner to minimize lung injury.     

Expression of laminin alpha1, alpha2, alpha4, and alpha5 chains, fibronectin, and tenascin-C in skeletal muscle of dystrophic 129ReJ dy/dy mice

The dy/dy mouse is an animal model for human merosin-negative congenital muscular dystrophy (CMD), which has been reported to have reduced or no expression of the basement membrane protein laminin alpha2. We here investigate various myogenic and nonmyogenic tissues of mature dy/dy and control 129ReJ mice histologically and for laminin alpha2 expression. In addition, expression patterns of laminin alpha1, alpha2, alpha4, and alpha5 chains, the interstitial proteins fibronectin and tenascin-C, and the adhesion molecules VCAM-1, ICAM-1, and alpha4 integrin were characterized in skeletal muscle of 1- and 7-day and mature (>6 weeks old) dy/dy and control 129ReJ mice. The laminin alpha2 chain remained detectable in myogenic tissues of dy/dy mice by immunofluorescence using two different monoclonal antibodies and by Northern blot analysis. However, laminin alpha2 expression was significantly reduced or not detectable in nonmyogenic tissues of dy/dy mice, including skin, lung, kidney, brain, thymus, and eye. Focal lesions were observed in mature skeletal muscle only, characterized by necrotic tissue, isolated VCAM-1- and ICAM-1-positive cells indicative of inflammatory processes, and regenerating muscle fibers surrounded by intense tenascin-C and fibronectin expression. In contrast to studies on human CMD muscle, laminin alpha1 was not detectable in either dy/dy or control skeletal muscle using immunofluorescence or Northern blot analysis. Immunofluorescence localized laminin alpha4 to basement membranes of blood vessels, the endoneurium of the intramuscular nerves, and the neuromuscular junction in skeletal muscle of 1- and 7-day-old dy/dy and control mice. In mature muscle, laminin alpha4 expression shifted to the perineurium of intramuscular nerves in both dy/dy and control mice. Furthermore, strong upregulation of laminin alpha4 in the basement membranes of blood vessels, the perineurium of intramuscular nerves, and of isolated regenerating muscle fibers in the dy/dy mice was apparent. Investigation of 1-day-old animals revealed expression of laminin alpha5 in skeletal muscle fiber basement membranes of dy/dy but not control animals. This difference between dy/dy and control animals was no longer apparent at 7 days after birth, indicating a temporary shift in expression pattern of laminin alpha5 in dy/dy animals. Analysis of the extracellular matrix components of 1- and 7-day-old dy/dy and control skeletal muscle revealed an early onset of the dystrophy, even before histopathological features of the disease were evident. Our data confirm the absence of laminin alpha1 chain in myogenic tissues of both dy/dy and control mice and suggest compensation for reduced laminin alpha2 in dy/dy skeletal muscle by laminin alpha4 and, in early development, also laminin alpha5. These results have significant ramifications in the diagnosis of human merosin-negative CMD.     

Structure, development and function of cytoskeletal elements in non-neuronal cells of the human eye

The cytoskeleton, of which the main components in the human eye are actin microfilaments, intermediate filaments and microtubules with their associated proteins, is essential for the normal growth, maturation, differentiation, integrity and function of its cells. These components interact with intra- and extracellular environment and each other, and their profile frequently changes during development, according to physiologic demands, and in various diseases. The ocular cytoskeleton is unique in many ways. A special pair of cytokeratins, CK 3 and 12, has apparently evolved only for the purposes of the corneal epithelium. However, other cytokeratins such as CK 4, 5, 14, and 19 are also important for the normal ocular surface epithelia, and other types may be acquired in keratinizing diseases. The intraocular tissues, which have a relatively simple cytoskeleton consisting mainly of vimentin and simple epithelial CK 8 and 18, differ in many details from extraocular ones. The iris and lens epithelium characteristically lack cytokeratins in adults, and the intraocular muscles all have a cytoskeletal profile of their own. The dilator of the iris contains vimentin, desmin and cytokeratins, being an example of triple intermediate filament expression, but the ciliary muscle lacks cytokeratin and the sphincter of the iris is devoid even of vimentin. Conversion from extraocular-type cytoskeletal profile occurs during fetal life. It seems that posttranslational modification of cytokeratins in the eye may also differ from that of extraocular tissues. So far, it has not been possible to reconcile the cytoskeletal profile of intraocular tissues with their specific functional demands, but many theories have been put forward. Systematic search for cytoskeletal elements has also revealed novel cell populations in the human eye. These include transitional cells of the cornea that may represent stem cells on migration, myofibroblasts of the scleral spur and juxtacanalicular tissue that may modulate aqueous outflow, and subepithelial matrix cells of the ciliary body and myofibroblasts of the choroid that may both participate in accommodation. In contrast to the structure and development of the ocular cytoskeleton, changes that take place in ocular disease have not been analysed systematically. Nevertheless, potentially meaningful changes have already been observed in corneal dystrophies (Meesmann's dystrophy, posterior polymorphous dystrophy and iridocorneal endothelial syndrome), degenerations (pterygium) and inflammatory diseases (Pseudomonas keratitis), in opacification of the lens (anterior subcapsular and secondary cataract), in diseases characterized by proliferation of the retinal pigment epithelium (macular degeneration and proliferative vitreoretinopathy), and in intraocular tumours (uveal melanoma). In particular, upregulation of alpha-smooth muscle actin seems to be a relatively general response typical of spreading and migrating corneal stromal and lens epithelial cells, trabecular cells and retinal pigment epithelial cells.     

Spatial compounding in 3D imaging of limbs

Catheter ablation for atrial fibrillation is based upon the critical mass of fibrillation hypothesis and aims to compartmentalize the atria by the creation of linear lesions, thereby reducing the amount of contiguous myocardium available for the propagation of multiple activation wavefronts. Early attempts at creating right atrial linear lesions with conventional catheter tip technology to treat patients with chronic and paroxysmal atrial fibrillation yielded disappointing results. Although more efficacious, the creation of extensive left atrial lesions has been associated with a high rate of thromboembolic stroke despite the administration of heparin and use of temperature feedback to control radiofrequency energy. Approaches to reduce the risk of stroke include: the creation of more continuous and effective right atrial lesions with linear array catheter technology and thereby reduce the requirement for left atrial ablation; the assessment of adjuvant pharmacological agents to inhibit platelet aggregation; guidance of radiofrequency energy delivery by intracardiac echo; assessment of transvenous cryotherapy as an alternative to radiofrequency energy in order to reduce endocardial disruption; and development of minimally invasive surgical approaches to left atrial epicardial ablation.     

内蒙古甲乌拉矿区节理发育分布规律与成矿关系的研究

甲乌拉矿是以铅锌银为主生产多年的老矿区,随着开采年限的延长,矿区320以上资源储量逐年减少,严重制约生产任务的完成。矿区在多年的生产过程中积累了大量的地质资料,前人也对矿区做了不少研究工作,也明确指出矿体呈脉状产于北西西、北西、北北西向断裂构造带中,但对节理方面的研究资料相对较少,尤其在节理分期、配套、构造应力分析等方面研究更少。本文通过对甲乌拉矿区野外考察和室内研究的综合分析,地层岩性的认识、构造背景的考察、岩石薄片的分析、200m中段节理产状的分析和整理、绘制节理玫瑰花图对节理进行分期、配套、应力分析等恢复矿区古应力场,在宏观构造的基础上进一步认识微观节理与矿体的形成、赋存等关系的研究,从而对矿区下一步地质找探矿方面提出一定的参考意见和措施。     

The subunit structure of myosin from skeletal muscle of the early chick embryo

The subunit composition and immunological properties of two types of myosins, the 3S and 6S myosin components, from skeletal muscle of early chick embryos were studied by SDS-acrylamide gel electrophoresis and immunodiffusion techniques. It was shown that the 6S myosin in the early embryonic stage was composed of two heavy chains and three kinds of light chains, as is well-known in the complete myosin molecule, having the same molecular weights and the same antigenicities as corresponding subunits of the myosin from adult chicken skeletal muscle. The heavy chain of 6S myosin was also reactive with the antibody against the heavy chain of cardiac myosin. The embryonic 3S myosin was shown to be composed of a heavy chain which was roughly the same in molecular weight but not the same in antigenicity as those of adult or embryonic 6S myosin. No light chains were detected either electrophoretically or immunologically in the 3S myosin component.     

Midbrain development induced by FGF8 in the chick embryo

Vertebrate midbrain development depends on an organizing centre located at the isthmus, a constriction in the embryonic mid/hindbrain region. Isthmic tissue grafts transform chick caudal forebrain into an ectopic midbrain that is the mirror image of the normal midbrain. Here we report that FGF8 protein has the same midbrain-inducing and polarizing effect as isthmic tissue. Moreover, FGF8 induces ectopic expression in the forebrain of genes normally expressed in the isthmus, suggesting that the ectopic midbrain forms under the influence of signals from a new 'isthmus-like' organizing centre induced in the forebrain. Because Fgf8 itself is expressed in the isthmus, our results identify FGF8 as an important signalling molecule in normal midbrain development.     

The morphological mechanism of the development of myosatellitocytes from the structural elements of the muscle fiber during increased functional activity of the skeletal muscles

Under increased muscular activity in some muscular fibers disintegration areas of myofibrillar apparatus has been revealed. Migration of myonuclei into these microregions starts the mechanism of their segregation due to plasmolemma produced from the reticulum sarcoplasmaticum and triad systems surface. After plasmolemma production in "sarcocytes" intensive development and differentiation of organellae occur. As a result of differentiation "sarcocytes" transform in to myosatellitocytes of type-2 and migrate under lamina externa muscular fibers. So, a hypothesis about formation of myogenic tissue's cellular phase from the myosymplastic one has been confirmed.