Biomedical subjects

S Thorsteinsdóttir

Publications and source records attributed to S Thorsteinsdóttir.

10 recordsLinked to original sources

PubMed1999

Early development of the myotome in the mouse.

The structure and development of the myotome has been extensively studied in birds and amphibians but few studies have systematically addressed its development in mammals. We have used a transgenic mouse carrying an nLacZ marker coupled to a myosin light chain 3F promoter to describe the structure of the developing mammalian myotome. Through studies of transgene expression pattern, coupled with immunohistochemistry for the muscle structural proteins desmin and slow myosin heavy chain we describe a gradient of maturity for the cells within the developing myotome. Our results show that the earliest myocytes of the mammalian myotome span the rostrocaudal extent of the somite and have single large nuclei which localise centrally within the myotome. Throughout the period of study the myotome is more mature ventrally than dorsally and cells comprising the medial aspect of the myotome are younger than those lying laterally. Immunohistochemistry for the earliest expressed muscle regulatory factor (myf-5) is used to define areas of the myotome contributing new myogenic cells. In the early myotome small, round, myf-5-expressing cells are found extensively within the dorsomedial aspect of the dermamyotome and also within the entire rostral and caudal dermamyotomal lips. They subsequently appear within the central zone of the myotome, adjacent to the medially curled rostral and caudal dermamyotomal lips, and there begin to elongate symmetrically. As the myotome enlarges, myf-5 expression is always restricted to the most medial aspect of the myotome, adjacent to the least mature myocytes, marking the site of addition of new myogenic cells. Together, these results allow development of a model of mammalian myotome formation where growth occurs medially by addition of new cells from both rostral and caudal dermamyotome lips, while more mature myocytes are displaced laterally. Furthermore, early myotomal myocytes differentiate in the absence of MyoD expression, unlike later myotomal myocytes. This, along with their distinct morphology, suggests these cells may form a separate lineage of pioneer myogenic cells.

Animals↗

PubMed1999

Expression of the alpha 6A integrin splice variant in developing mouse embryonic stem cell aggregates and correlation with cardiac muscle differentiation.

Mouse embryonic stem (ES) cells grown in aggregates give rise to several different cell types, including cardiac muscle. Given the lack of cardiac muscle cell lines, ES cells can be a useful tool in the study of cardiac muscle differentiation. The laminin-binding integrin alpha 6 beta 1 exists in two different splice variant forms of the alpha chain (alpha 6A and alpha 6B), the alpha 6A form having been implicated as possibly playing a role in cardiac muscle development, based on its distribution pattern [4, 53]. In this study we characterise the ES cell model system in terms of the expression of the two different alpha 6 splice variants. We correlate their expression with that of muscle markers and the transcription factor GATA-4, using the reverse transcription-polymerase chain reaction (RT-PCR). We confirm that alpha 6B is constitutively expressed by ES cells. In contrast, alpha 6A expression appears later and overlaps in time with a period when the muscle marker myosin light chain-2V (MLC-2V) is expressed, but no MyoD is present, which indicates the presence of cardiac muscle cells in the aggregates. We further show that GATA-4 is present at the same time. Culturing the aggregates under conditions that stimulate (transforming growth factor beta 1 supplement) or inhibit (TGF beta 1 plus 10(-9) M retinoic acid supplement) cardiac muscle differentiation does not lead to any qualitative differences in the timing of expression of these genes, but quantitative changes cannot be excluded. The TGF beta 1 supplement does, however, lead to a relatively greater expression of alpha 6A compared to alpha 6B than the TGF beta 1 plus 10(-9) M RA supplement after 6 days in culture, suggesting that alpha 6A expression is favoured under conditions that stimulate cardiac muscle differentiation. The switch towards alpha 6A expression in ES cell aggregates is paralleled by expression of the binding receptor for TGF beta (T beta RII). Stable expression of a mutated (dominant negative) T beta RII in ES cells, however, still resulted in (TGF beta-independent) upregulation of alpha 6A, demonstrating that these events were not causally related and that parallel or alternative regulatory pathways exist. The initial characterisation of differentiating ES cell aggregates in terms of alpha 6A integrin subunit expression suggests that this model system could be a valuable tool in the study of the role of the alpha 6A beta 1 integrin in cardiac muscle differentiation.

Animals↗

PubMed1999

Partial rescue of epithelial phenotype in integrin beta4 null mice by a keratin-5 promoter driven human integrin beta4 transgene.

Integrin beta4 null mice exhibit extensive epidermal detachment, reminiscent of the human skin blistering disease junctional epidermolysis bullosa associated with pyloric atresia. Hemidesmosomes, the stable adhesion structures of squamous epithelia, are not formed in the absence of alpha6beta4. Null mutant mice die shortly after birth, but apart from their striking epithelial phenotype, no obvious developmental defects have been observed. To elucidate the cause of death in these mice, we generated transgenic mice with a heterologous construct consisting of the squamous epithelial-specific keratin-5 promoter and a human integrin beta4 subunit cDNA. The transgene was not expressed in the presence of endogenous beta4, probably as a result of competition for a limited pool of alpha6 subunits. In a beta4 null background, however, the transgene was expressed, and its expression pattern followed that of squamous epithelial-specific keratins. These rescued pups appeared healthy and ultrastructural analysis revealed that the interspecies heterodimer alpha6(mouse)/beta4(human) was sufficient to trigger the assembly of hemidesmosomes. After a variable period of up to 48 hours after birth these animals began to exhibit haemorrhages at the plantar and palmar areas. We observed the formation of small blisters and found that the transgene was not detectably expressed in this region, which is devoid of hair follicles. The rescued neonates became increasingly cyanotic and died soon after the onset of this phenomenon. We performed a developmental study of the expression of beta4 in the complete respiratory tract, but we found no correlation between the spatiotemporal distribution of beta4 and the onset of the respiratory insufficiency. It became clear, however, that there was a gradual detachment of squamous epithelia in the oral and nasal cavities which led to obstruction of the respiratory tract, suggesting that in beta4 null and rescued mice, neonatal death was a direct consequence of decreased adhesion properties of hairless squamous epithelia, rather than a developmental defect of the lungs.

Animals↗

PubMed1997

Spatial and temporal expression of the beta1D integrin during mouse development.

The beta1D protein is a recently characterized isoform of the integrin beta1 subunit that is present in cardiac and skeletal muscles. In this study, we have examined the expression of beta1D in different types of skeletal muscle and in cardiac muscle and studied its distribution during mouse development, using new monoclonal antibodies specific for beta1D. Immunoprecipitation studies revealed that, while beta1A is strongly expressed in proliferating C2C12 myoblasts, beta1D is only expressed after their differentiation to myotubes. In these myotubes, beta1D is associated with different alpha subunits, namely alpha3A, alpha5, alpha7A, or alpha7B. Initially, during embryogenesis, the alpha1A subunit is the only beta1 variant expressed in skeletal and cardiac muscle. The beta1D subunit is first detected in skeletal muscle at E17.5, whereas in cardiac muscle its expression begins around the time of birth. Later the expression of beta1A in skeletal and cardiac muscle becomes restricted to capillary cells, whereas beta1D eventually becomes the only variant expressed in adult cardiac and skeletal muscle cells. The switch from the beta1A to the beta1D subunit in cardiac muscle cells coincides with the expression of alpha7. In adults there is a distinct concentration of beta1D at the myotendinous junctions of muscle fibers and at costameres in both cardiac and skeletal muscle. In addition, beta1D is present at intercalated discs in cardiac muscle and at neuromuscular junctions in skeletal muscle cells. The amount of beta1D in different types of skeletal muscle (fast, slow, and mixed-type) was similar, but cardiac muscle expressed almost five times as much of this protein. We suggest that beta1D plays a role in the maintenance of the cytoarchitecture of mature muscle and in the functional integrity of the muscle cells.

Animals↗

PubMed1995

Expression patterns of laminin receptor splice variants alpha 6A beta 1 and alpha 6B beta 1 suggest different roles in mouse development.

The alpha 6 beta 1 integrin is a receptor for laminins and is present from early stages of mouse embryogenesis. In the present study we determined the temporal and spatial expression of the two cytoplasmic splice variants of the alpha 6 integrin subunit, alpha 6A and alpha 6B, in the early- and mid-gestation mouse postimplantation embryo using RT-PCR, in situ hybridization, and immunofluorescence. Our results show that alpha 6B is present in the embryo at all stages studied and is expressed before alpha 6A. alpha 6A expression begins in 8.5 day p.c. embryos and is initially exclusively localized to the developing heart. In 8.5 (and 9.5) day p.c. embryos alpha 6A mRNA and protein are present in a gradient in the myocardium of the heart tube from strongest expression in the sinus venosus and in the common atrial chamber to a weakening expression along the ventricle and bulbus cordis. In 10.5 day p.c. embryos this gradient is less evident and in 12.5 day p.c. embryos alpha 6A mRNA and protein are present in comparable amounts between atria and ventricles. Neither alpha 6A nor alpha 6B is present in endocardial cushion tissue. By day 12.5 p.c. alpha 6A expression is also present in the developing epidermis, dental primordia, lens, gonads, and in a few epithelia such as those of the digestive tract. alpha 6B expression is always much more widespread than alpha 6A expression. For example, only alpha 6B is present in the myotome of the somites of 9.5 day p.c. embryos, in the developing central and peripheral nervous systems, and in the nephrogenic system at all stages studied, except after the differentiation of the gonads when alpha 6A is also present. Furthermore, alpha 6B is the only splice variant present on endothelial cells. We also examined the distribution of the beta 4 integrin subunit to determine whether the alpha 6 beta 4 integrin was present during these stages of development. Beta 4 protein was absent in early postimplantation stages but was present in the epidermis and digestive tract of 12.5 day p.c. embryos. These results show a differential distribution of alpha 6A and alpha 6B during mouse development and thus strongly suggest a different function of these splice variants during embryogenesis. Our results point to a possible role for the alpha 6A beta 1 integrin in the development of the myocardium of the developing heart, but not in the migration of endocardial cushion cells, while alpha 6B beta 1 could be important in the developing nephrogenic and nervous systems.

Alternative Splicing↗

PubMed1993

Variants of the alpha 6 beta 1 laminin receptor in early murine development: distribution, molecular cloning and chromosomal localization of the mouse integrin alpha 6 subunit.

Laminin (A:B1:B2) is a major component of the first basement membrane to appear in the developing mouse embryo. Its effects on morphogenesis and differentiation are mediated by interaction with cell surface receptors that are members of the integrin family. We have studied the expression of the alpha 6 subunit of murine alpha 6 beta 1 and its ligand, laminin, in preimplantation mouse embryos, embryo outgrowths and in embryonic stem (ES) cells and embryonal carcinoma (EC) cells. The alpha 6 subunit is present in the oocyte and throughout preimplantation development. Laminin A chain appears later than alpha 6 and has a more restricted distribution until the late blastocyst stage. alpha 6 beta 1 is strongly expressed in ES and EC cells; the levels of mRNA expression are not altered by differentiation. Molecular cloning of cDNA for the murine integrin alpha 6 subunit from a mammary gland lambda gt11 library showed, as in man, an open reading frame encoding two variants of alpha 6, alpha 6A and alpha 6B. The identity of the alpha 6 amino acid sequence to that in man and chicken is 93% and 73%, respectively. The gene for murine alpha 6 was mapped to chromosome 2. While undifferentiated ES and EC cells express only alpha 6B, alpha 6A is co-expressed in ES cells after differentiation is induced by retinoic acid. alpha 6B is also the only variant expressed in blastocyst stage embryos, but when blastocysts have grown out in culture both alpha 6A and alpha 6B are expressed reflecting the results in the cell lines. We suggest that the deposition of laminin in the embryo is a receptor-mediated process and that the shift in the expression of the variants, as the inner cell mass forms its first differentiated progeny, reflects a change in functional properties.

Amino Acid Sequence↗

PubMed1992

Basement membrane and fibronectin matrix are distinct entities in the developing mouse blastocyst.

Immunocytochemical techniques were used to study the distribution of fibronectin, type IV collagen (collagen-IV), and laminin in four different stages of mouse blastocyst development. Immunoreactivity for collagen-IV and laminin is present in a granular pattern inside the inner cell mass (ICM) cells in stage 1 blastocysts, while these blastocysts are negative for fibronectin. Fibronectin immunoreactivity appears extracellularly under the trophectoderm (TE) in stage 2 blastocysts, in the form of homogeneously distributed dots, and/or fibrils located preferentially close to cell boundaries. It is followed by the appearance of both collagen-IV and laminin immunoreactivity in patches on the basal side of the TE in stage 3 blastocysts. These patches are initially localized under the central region of TE cells, thus in a location clearly different from that of fibronectin-positive fibrils. As development proceeds the collagen-IV- and laminin-positive patches become larger, covering, by stage 4, an extensive portion of the inner lining of the blastocoel. Fibronectin-positive material is still present in a fibrillar form in stage 3 blastocysts, but is generally reduced to thin strands by stage 4. These results indicate that fibronectin is independent of the mouse blastocyst basement membrane, but may play a transient role in cell adhesion during its deposition. In addition, the results suggest that the ICM plays a major role in the production of collagen-IV and laminin, while the basal surface of TE cells is the primary site of basement membrane assembly.

Animals↗

PubMed1990

Immunocytochemical study of tissue distribution and hormonal control of chondroitin-, dermatan- and keratan sulfates from rodent uterus.

The distribution patterns of rat and mouse uterine glycosaminoglycans (GAGs), as well as their modulation by estradiol (E2) and/or progesterone (P), were investigated using monoclonal antibodies (MABs) directed against chondroitin- (CS)/dermatan sulfates (DS), keratan sulfate (KS) and a trophoblast GAG. The localization of GAGs in relation to collagens (I, IV and VI) and fibronectin was also analyzed. We found that uterine GAGs are differentially distributed in the endometrium and myometrium, in a pattern that is species-related. CS-containing proteoglycans (PGs) occur between collagen bundles and fibroblasts, at the periphery of the latter, and in basement membrane zones (BMZs), in a pattern resembling that of collagen VI. BMZs contain preferentially CS-PGs bearing 4-sulfated disaccharides adjacent to the core protein. DS-PGs are mostly associated with collagen bundles. E2 and/or P elicit distinct modifications on the above described pattern, which are also species-related. The simultaneous administration of E2 and P changes the prevalent sulfation of the disaccharides adjacent to the core protein of stromal CS-PGs. In the mouse, an unsulfated intracellular epitope appears following E2 (or E2P) administration, mostly in epithelial cells. In the rat, KS and the trophoblast GAG are E2-dependent and down-regulated by P. The functional significance of the hormone-induced GAG changes, namely the possible role of the E2-dependent KS in implantation, are discussed.

Animals↗

PubMed1988

Reevaluation of fibronectin-collagen interactions in tissues: an immunocytochemical and immunochemical study.

We studied the distribution of fibronectin (FN) in rat uterus, rat tail tendon, and rooster comb, using LM and EM immunocytochemistry. Special attention was paid to the interaction of FN with collagen (COL). Various labeling protocols and dot-blot experiments were performed to confirm the results. Under conditions of labeling specificity, FN distribution over native COL fibrils was usually sparse, especially when these were organized into thick bundles. No labeling was observed over section surfaces of COL fibrils when postembedding methods were used, which indicates that no FN is present within these fibrils. Under conditions in which exogenous FN could react with tissues, e.g., when preincubation with normal serum for background blocking was performed, artifactual staining appeared over COL. Such a reaction also occurred when anti-FN antiserum completely blocked by liquid-phase adsorption was used. Therefore, the FN present in soluble FN-anti-FN immune complexes must have still been able to react with COL. The artifactual labeling was, in all cases, almost exclusively localized on the section surfaces of COL fibrils. These results suggest that FN has a very low affinity for the surface of native COL fibrils.

Animals↗

PubMed1985

Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2.

We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.

Anticodon↗