Extracellular matrix fluctuations during early embryogenesis

Extracellular matrix (ECM) movements and rearrangements were studied in avian embryos during early stages of development. We show that the ECM moves as a composite material, whereby distinct molecular components as well as spatially separated layers exhibit similar displacements. Using scanning wide field and confocal microscopy we show that the velocity field of ECM displacement is smooth in space and that ECM movements are correlated even at locations separated by several hundred micrometers. Velocity vectors, however, strongly fluctuate in time. The autocorrelation time of the velocity fluctuations is less than a minute. Suppression of the fluctuations yields a persistent movement pattern that is shared among embryos at equivalent stages of development. The high resolution of the velocity fields allows a detailed spatio-temporal characterization of important morphogenetic processes, especially tissue dynamics surrounding the embryonic organizer (Hensen's node).     

Spatial and temporal patterns of cell division during early Xenopus embryogenesis

We describe the spatial and temporal patterns of cell division in the early Xenopus embryo, concentrating on the period between the midblastula transition and the early tailbud stage. Mitotic cells were identified using an antibody recognising phosphorylated histone H3. At least four observations are of interest. First, axial mesodermal cells, including prospective notochord, stop dividing after involution and may not divide thereafter. Second, cell division is more pronounced in the neural plate than in nonneural ectoderm, and the pattern of cell division becomes further refined as neurogenesis proceeds. Third, cells in the cement gland cease proliferation completely as they begin to accumulate pigment. Finally, the precursors of peripheral sensory organs such as the ear and olfactory placode undergo active cell proliferation when they arise from the sensorial layer of the ectoderm. These observations and others should provide a platform to study the relationship between the regulation of developmental processes and the cell cycle during Xenopus embryogenesis.     

Spatial and temporal patterns of ERK signaling during mouse embryogenesis

Signaling between tissues is essential to form the complex, three-dimensional organization of an embryo. Because many receptor tyrosine kinases signal through the RAS-MAPK pathway, phosphorylated ERK can be used as an indicator of when and where signaling is active during development. Using whole-mount immunohistochemistry with antibodies specific to phosphorylated ERK1 and ERK2, we analyzed the location, timing, distribution, duration and intensity of ERK signaling during mouse embryogenesis (5-10.5 days postcoitum). Spatial and temporal domains of ERK activation were discrete with well-defined boundaries, indicating specific regulation of signaling in vivo. Prominent, sustained domains of ERK activation were seen in the ectoplacental cone, extra-embryonic ectoderm, limb buds, branchial arches, frontonasal process, forebrain, midbrain-hindbrain boundary, tailbud, foregut and liver. Transient activation was seen in neural crest, peripheral nervous system, nascent blood vessels, and anlagen of the eye, ear and heart. In the contiguous domains of ERK signaling, phospho-ERK staining was cytoplasmic with no sign of nuclear translocation. With few exceptions, the strongest domains of ERK activation correlated with regions of known or suspected fibroblast growth factor (FGF) signaling, and brief incubation with an inhibitor of the fibroblast growth factor receptor (FGFR) specifically diminished the phospho-ERK staining in these regions. Although many domains of ERK activation were FGFR-dependent, not all domains of FGF signaling were phospho-ERK positive. These studies identify key domains of sustained ERK signaling in the intact mouse embryo, give significant insight into the regulation of this signaling in vivo and pinpoint regions where downstream target genes can be sought.     

Spatial and temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during avian embryogenesis

A-CAM (adherens-junction-specific cell adhesion molecule) is a calcium-dependent adhesion molecule which is associated with intercellular adherens junctions in various tissues (Volk & Geiger, 1986, J. Cell Biol. 103, 1441-1450 and 1451-1464). In the present report, we have investigated the distribution of A-CAM during avian morphogenesis by immunofluorescence microscopy and immunoblotting. A-CAM appeared at the onset of gastrulation on developing mesodermal and endodermal cells and was then expressed on tissues derived from the three primary germ layers. During embryonic life, A-CAM was constitutively expressed in a number of tissues including the central and peripheral nervous system, myocardium, muscles, notochord, skin and lens whereas it was found transiently in many tissues ranging from the nephritic tubules and the endoderm of visceral arches to ectodermal placodes. In the adult, in addition to the nervous system, A-CAM was restricted to the skin, lens, heart and testis, and exhibited an apparent molecular weight higher than the one found in the embryo. The prevalence and cell-surface modulation of A-CAM could frequently be correlated with morphogenetic events such as mesenchyme condensation into epithelia or cell clusters (e.g. formation of the somitic epithelium, kidney tubules and peripheral ganglia), dissociation of epithelia (e.g. dissociation of the somitic epithelium and segregation of neural crest from the neural tube), separation of cell populations (e.g. fibroblasts and myotubes in the heart) and reorganizations of epithelia (e.g. neurulation). In addition, using electron microscopy, the expression of A-CAM on the surface of aggregating and separating cells could be correlated with the formation and disappearance of adherens junctions. This precisely scheduled control of A-CAM correlated with early morphogenetic events during embryogenesis suggests that this CAM could play a crucial role in these processes.     

The temporal pattern of interphase prolongation and nuclear activities during early embryogenesis in teleostei

Summary Temporal relationship between the beginning of the increase of interphase duration, RNA synthesis in the nuclei and morphogenetic function of nuclei have been studied using dimensionless criteria of development duration. Experiments were performed in the trout (Salmo trutta m.fario andSalmo irideus), the whitefish (Coregonus lavaretus), the pike (Esox lucius) and the carp (Cyprinus caprio). In all these species interphase begins to lengthen approximately at the same developmental stages. The onset of morphogenetic nuclear function in the pike and whitefish coincides with the onset of interphase prolongation; in the carp RNA synthesis in the nuclei and morphogenetic nuclear function were revealed by 2 ₀ earlier than onset of interphase prolongation, and in the trout, by 5 and 13 ₀ later, respectively. Thus, a temporal disconnection between the onset of interphase prolongation, RNA synthesis in the nuclei and morphogenetic nuclear function was established for the first time. It can be suggested that the character of temporal relationship between these processes depends on the relative quantity of yolk in the eggs.

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Zusammenfassung In der Embryogenese von Forelle, Weißfisch, Hecht und Karpfen ist untersucht worden, welche zeitlichen Beziehungen bestehen zwischen dem Beginn der Zunahme der Interphasedauer, der RNS-Synthese in den Kernen und der morphogenetischen Funktion der Kerne. Bei allen genannten Arten verlängert sich die Interphase etwa in dem gleichen Entwicklungsstadium. Beim Hecht und beim Weißfisch beginnt die morphogenetische Funktion der Kerne gleichzeitig mit der Verlängerung der Interphasedauer; beim Karpfen setzen RNS-Synthese und morphogenetische Funktion der Kerne zwei Zeiteinheiten ( ₀) früher ein als der Beginn der Interphaseverlängerung, bei der Forelle dagegen 5 bzw. 13 Zeiteinheiten später. Damit ist erstmals gezeigt worden, daß kein zeitlicher Zusammenhang besteht zwischen dem Beginn von Interphaseverlängerung, RNS-Synthese in den Kernen und morphogenetischer Funktion der Kerne. Vermutlich hängt die zeitliche Beziehung zwischen diesen Vorgängen vom relativen Dottergehalt der Eier ab.

     

Sea urchin arylsulfatase,an extracellular matrix component,is involved in gastrulation during embryogenesis

Arylsulfatases (Arses) have been regarded as lysosomal enzymes because of their hydrolytic activities on synthetic aromatic substrates and their lysosomal localization of their enzymatic activities. Using sea urchin embryos, we previously demonstrated that the bulk of Hemicentrotus Ars (HpArs) does not exhibit enzyme activity and is located on the apical surface of the epithelial cells co-localizing with sulfated polysaccharides. Here we show that HpArs strongly binds to sulfated polysaccharides and that repression of the synthesis by HpArs-morpholino results in retardation of gastrulation in the sea urchin embryo. Accumulation of HpArs protein and sulfated polysaccharides on the apical surface of the epithelial cells in sea urchin larvae is repressed by treatment with β-aminopropionitrile (BAPN), suggesting that deposition of HpArs and sulfated polysaccharides is dependent on the crosslinking of proteins such as collagen-like molecules. We suggest that HpArs functions by binding to components of the extracellular matrix.     

Spatial and temporal expression patterns directed by theAgrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot

We have analysed the patterns of expression of a gene encoding -glucuronidase (GUS) fused to the promoter of theAgrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot,Daucus carota L. Gene expression was monitored by a histochemical assay of -glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.     

Arabinogalactan-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv.

The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of arabinogalactan-protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation.     

A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts

Background

Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.

Results

Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.

Conclusions

microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.     

Spatial interrelationships between proteoglycans and extracellular matrix proteins in cell cultures

Immunolabelling of cultured cells for chondroitin-sulfate proteoglycan (CSPG), in conjunction with antibodies to fibronectin, collagen and laminin, revealed the spatial interrelationships between the different matrix components. CSPG was organized in two major forms. Fibronectin-independent dotted patterns of CSPG were detected on the substrate and cell surfaces at early stages after plating. At later stages, however, significant overlapping was found between the two extracellular matrix components. Immunoelectron microscopic examination indicated that the CSPG was organized as granules of varying sizes which were associated with the cell surface, the substrate, or with the periphery of the fibronectin network.     

Structural changes during early embryogenesis in wheat pollen

Summary We have made a detailed cytological examination of the development of wheat embryoids, monitoring their initial divisions from two to ten cells by both light and electron microscopy. According to our observations the first embryogenic division is symmetrical. After the androgenesis induction treatment, there is a decrease in ribosome population with cells that have inactive nucleoli made up almost exclusively of a dense fibrillar component. This population is restored after initial embryogenic divisions. During the initial divisions the embryogenic pollen grains do not appear to change in size and the pollen wall remains intact. The exine undergoes no modification but the intine thickens, and we have observed that the thickness of the intine can be used as a cytological marker of androgenesis. The walls separating the cells obtained after embryogenic division contained numerous plasmodesmata. The beginnings of embryo polarization and cell differentiation could be made out in the very early pollen embryoids.     

Adapted Boolean network models for extracellular matrix formation

Background  

Due to the rapid data accumulation on pathogenesis and progression of chronic inflammation, there is an increasing demand for approaches to analyse the underlying regulatory networks. For example, rheumatoid arthritis (RA) is a chronic inflammatory disease, characterised by joint destruction and perpetuated by activated synovial fibroblasts (SFB). These abnormally express and/or secrete pro-inflammatory cytokines, collagens causing joint fibrosis, or tissue-degrading enzymes resulting in destruction of the extra-cellular matrix (ECM).     

Differential temporal and spatial expression of mRNA encoding extracellular matrix components in decidua during the peri-implantation period.

Changes in the temporal and spatial patterns of expression of mRNA encoding uterine extracellular matrix (ECM) proteins were determined during the peri-implantation period. Northern blot hybridization of cDNAs corresponding to laminin (LM) B1, LM B2, entactin, fibronectin, collagen (CL) type IV alpha 1, and CL IV alpha 2 was performed on RNA extracted from either whole mouse uteri or endometrial explants between Day 4, i.e., the day of implantation, and Day 7 of pregnancy, when the decidual response is well established. These analyses revealed a dramatic increase in LM B2, CL IV alpha 1, and CL IV alpha 2 mRNA expression by Day 7 of pregnancy. Relative levels of the mRNA encoding other ECM components, including LM B1, were not altered when compared to changes in the relative level of expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. The differential expression of the B chains of LM appeared to be limited to the stromal cells of the endometrium. In situ hybridization of uterine sections with cRNA probes corresponding to LM B1, LM B2, and CL IV alpha 1 demonstrated that LM B1 was expressed temporally in high amounts in the primary decidual zones (PDZ) and persisted throughout PDZ degeneration. LM B2 mRNA was expressed in both primary and secondary decidual zones and persisted through Day 8 of pregnancy. CL IV alpha 1 mRNA expression mimicked that of LM B2. Oviduct ligation on Day 2 of pregnancy was used to prevent embryo transport to one uterine horn, whereas decidualization and embryo implantation were permitted in the contralateral horn. This experiment demonstrated that the increases in uterine ECM mRNA expression were not due solely to the changing hormonal milieu of the uterus. ECM components, including CL IV, have been shown to bind growth factors such as transforming growth factor-beta (TGF-beta) in an insoluble but biologically active form. The remarkable similarity between the pattern of CL IV and LM B2 expression and previously reported TGF-beta deposition (Tamada et al., Mol Endocrinol 1990; 4:965-972) prompted examination of the effects of this growth factor on blastocyst development in vitro. TGF-beta 1 was tested for its ability to alter embryo outgrowth on LM-coated tissue culture surfaces; however, significant differences in the rate or extent of outgrowth in the presence of TGF-beta were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

Background  

Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow.     

Differential expression of insulin-like growth factors in the uterine epithelium and extracellular matrix during early pregnancy

We have studied the simultaneous expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) in the uterine epithelium and extracellular matrix during the time of trophoblast attachment and implantation. These studies reveal that IGF-I and IGF-II display different spatial and temporal patterns of expression during early pregnancy, and suggest a role for them in the process of attachment and implantation. Specifically, IGF-I is strongly expressed in the basal lamina which is the site of trophoblast invasion into the maternal stroma, and also in the apical epithelium, the site of initial trophoblast attachment. IGF-II is expressed to a lesser extent in the basal lamina, lateral plasma membranes and apical epithelium on day 3 but is only prominent apically at the time of implantation, suggesting a role in attachment.