Development Invivo Research Articles

Changes in IP3 Metabolism During Skeletal Muscle Development In Vivo and In Vitro

We have investigated whether IP3 metabolism presents particular changes during critical stages of muscle development. With this aim, we have measured IP3 formation through phospholipase C activity, IP3 removal through IP3 5-phosphatase and IP3 3-kinase activities, as well as IP3 mass, during myogenesis in vivo and in vitro. In developing rat skeletal muscle, both IP3 3-kinase and 5-phosphatase activities were relatively constant from embryonary day 15, the earliest age studied, to postnatal day 10; 5-phosphatase decreased upon further development. A transient, major increase in phospholipase C activity was evident at embryonary day 18 while a non-significant increase in IP3 mass was detected at this embrionary age. In rat skeletal muscle in primary culture, all enzyme activities as well as the mass of IP3 increased significantly in myotubes compared to myoblasts. Myotubes incubated with calcitonin gene-related peptide, responded with a transient increase in IP3 mass after 2 to 10 sec; the CGRP-induced increase being completely blocked by U-73122, a phospholipase C inhibitor. Furthermore, IP3 mass increased within 1 hr after exposure to differentiating agents of both RCMH cells, a line derived from normal human skeletal muscle, and C2C12 cells. These results indicate that changes in IP3 metabolism can be correlated to critical stages of muscle development and differentiation, suggesting a possible role for IP3 in these processes.

Interactions Between c-kit and Stem Cell Factor Are Not Required for B-Cell Development In Vivo

AbstractThe receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit− HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kitnull mutant (W/W ) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2−/− mice. Surprisingly, transferred c-kit− cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit− HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.

Recombinant human bone morphogenetic protein (rhBMP) induced heterotopic bone development in vivo and in vitro.

In vivo, recombinant human bone morphogenetic protein (rhBMP-2) with deactivated bone matrix as a carrier, implanted in muscle in adult rates, induced development of heterotopic bone, including bone marrow. The volume of bone was proportional to the dose of rhBMP-2 in a range of 0.2-150 microg. In vitro, in response to 50-microgram dose range, subcutis- and brain-derived outgrowths differentiated into loosely woven connective tissues composed of spindle-shaped fibroblasts, adipocytes, and cartilage. Muscle-derived connective tissues cultivated first in culture media supplemented with 50 microgram of rhBMP-2 for 72 hr, then enclosed in a diffusion chamber, and immediately transplanted into a rectus abdominous muscle pouch in an autogenic rat for 28 days, induced cartilage development on the inside and transmembrane hetertoptic bone development including bone marrow on the outside. These experiments are interpreted to show that muscle derived connective tissue cells have the competence of embryonic cells to develop de novo in response to BMP in postfetal life.

B Lymphocyte Development and Transcription Regulation in Vivo

Publisher Summary The targeted mutation and ectopic or forced expression assays in vivo have helped identify genes that encode factors necessary for normal B cell development. In somewhat more depth, spatial aspects of transcription regulation are outlined in this chapter, as these are crucial to the understanding of how well-balanced specific changes in gene expression are achieved in vivo . The current knowledge of B cell development in the fetal, neonatal, young adult, and older mouse is reviewed, with an emphasis on the changes in gene expression patterns. The advancement in understanding the marrow organ as inductive microenvironment for postnatal B cell development is also highlighted. The chapter also reviews how the experiments on transcription regulation enrich the insight in pathways of B cell genesis. Further identification of genes disturbed in their expression, including those encoding cell cycle and death regulators, will give insight in genetic programs operating in hemopoiesis and B cell development. Novel genetic tools in vivo allows a follow-up of the effect of mutations in healthier animals. Transcription factors necessary for B cell development may be tested further for the ability to drive the differentiation pathways by ectopic expression in suitable cell types, for example, in hemopoietic stem and progenitor cells, by using the control region of the CD34 gene. The current information on the phenotype of mice with mutations in transcription factors indicates that much valuable information may still be retrieved from them. Together with the growing insight in surface receptor engagement and signaling pathways, an integrative vision of B cell development in vivo is emerging.

Maternal dietary glucose-lipid interactions modulate embryological development in vivo and in embryo culture.

To examine the dietary interrelationships of glucose and lipid on embryological growth and development, two levels of glucose (0% and 24%) and two types of lipid--soybean oil (SBO; 44.5% 18:2[n-6]) or oleic acid (OL; 6.7% 18:2[n-6])--were fed to pregnant rat dams until Day 12.5 of gestation, when developing embryos and maternal tissues were collected for analysis. In addition, Day 9.5 embryos were obtained from the dietary treatment groups and cultured in vitro until Day 12.5 to ascertain whether or not the embryos showed developmental changes in response to dietary treatment in the absence of maternal and placental factors. Differences in energy intake and macronutrient composition explained differences in early placental weight; macronutrient composition, but not energy intake, accounted for differences in weight of in vivo embryos and in morphological scores of cultured embryos. With food intake as a covariate in the statistical analysis, the results showed that only the level of maternal dietary glucose influenced the number of live embryos and resorptions per litter. Both the level of carbohydrate and the type of lipid in the maternal diet, however, independently and interactively affected both embryonic growth and development in vivo and in culture. To evaluate the biological significance of this dietary interaction, concentrations of essential fatty acids in the embryonic and maternal tissues were measured. A striking result of this study was the rapidity of the diet-induced changes in tissue fatty acid composition. By gestational Day 12.5, the lack of glucose in the OL-based diet was associated with a raised 18:2 (n-6)/20:4 (n-6) ratio and a lowered 20:4 (n-6) content in the embryo and the maternal liver. We suggest that a carbohydrate-free maternal diet with apparently adequate levels of essential fatty acid can contribute to embryonic growth retardation both in vivo and in vitro by perturbing embryonic essential fatty acid metabolism.

A developmental study of lactate dehydrogenase isozyme and aspartate aminotransferase activity in organotypic rat hippocampal slice cultures and primary cultures of mouse neocortical and cerebellar neurons

The development of enzyme activity and isozyme distribution of lactate dehydrogenase (LDH) was studied in murine organotypic hippocampal slice cultures and dissociated cultures of neocortical neurons and cerebellar granule cells and compared with that of the respective brain regions invivo. In the hippocampal slice cultures and the hippocampus invivo, the activity of aspartate aminotransferase (AAT) was also measured. During development in culture the specific activity of LDH increased in all types of cultures reaching values similar to that found in the corresponding brain areas invivo. However, significant differences in the isozyme distribution were observed between the preparations in vitro and invivo. During development invivo, the LDH isozyme pattern changed from a preferential M-subunit composition to a preferential H-subunit composition regardless of the brain area. This shift was not observed in the respective cultures where the M4-isozyme prevailed at all culture periods examined accounting for 30–45% of the total LDH activity. The cultured cerebellar granule cells did not express the H4-isozyme at all, while in the hippocampal slice cultures and the cultured neo-cortical neurons this isozyme accounted for about 5% of the total LDH activity. The activity of AAT in the hippocampal organotypic slice cultures increased considerably during the culture period in parallel with the increase in AAT activity during postnatal development of hippocampus invivo. The activity of AAT in the slice cultures was, however, consistently lower than the corresponding activity invivo. The results show that perinatal hippocampal tissue and neurons from neocortex or cerebellum retain their immature LDH-isoenzyme distribution when grown as organotypic slice cultures or dissociated cell cultures, respectively. This lack of shift from the M4 to the H4 LDH isozyme which is typical of tissues relying on aerobic glycolysis has several implications but does not apparently interfere with the otherwise normal differentiation of morphological, biochemical and electrophysiological characteristics of these preparations.

Reconstitution of Hair Follicle Development In Vivo: Determination of Follicle Formation, Hair Growth, and Hair Quality by Dermal Cells

Combinations of cultured and uncultured epidermal and dermal cell preparations from newborn and perinatal mice were grafted onto the backs of athymic nude mouse hosts to elucidate the cellular requirements for skin appendage formation. All epidermal populations studied, including a total epidermal keratinocyte preparation from trypsin-split skin, developing hair follicle buds isolated from epidermis, and preformed hair follicles isolated from dermis, make haired skin when grafted with fresh dermal cells. Only pre-formed hair follicles produce haired skin on grafts without an additional dermal component. Hair follicle buds grafted alone or with cultured dermal cells will reconstitute skin but without appendage formation. Thus, cells or factors present in fresh, but not cultured, dermal cells are essential for supporting hair growth from budding follicles, whereas more developed (pre-formed) follicles appear to contain all the necessary components for hair formation. Dissociation of isolated hair follicles by trypsin/ethylenediaminetetraacetic acid prior to grafting is permissive for hair growth, suggesting that follicle cells can be re-induced or reassociate in vivo. Dermal papilla cells, microdissected from rat vibrissal follicles and cultured for up to 14 passages, stimulate hair growth from follicle buds and influence the quality of hair growth from pre-formed hair follicles. Thus, dermal papilla cells maintain inductive capacity in culture and contribute to the reconstituted skin. This reconstitution model should be useful for identifying cell populations within the hair follicle compartment necessary for hair growth and for examining the effects of specific gene products on hair follicle growth and development in vivo.

Embryologic development in vivo and in vitro of the dark cell region of the mammalian crista ampullaris.

The area comprising the dark cells around vestibular organs was analysed with regard to embryologic development and maturation from the 13th gestational day (otocyst stage) (CBA/CBA mouse) to birth at the 21st gestational day when the organ reveals a morphologic maturation of inner ear gross structure and as also cytodifferentiation of vestibular hair cells. The development occurred parallel in vivo and in vitro. Electron-optically dense pigments below the dark cell region were first identified on the 15th gestational day. On the 16th gestational day the future dark cells have their mitochondria accumulated to the infranuclear region of the cell. From the 17th day onward, intercellular spaces develop close to the basal membrane, forming at birth a meshwork of interdigitations. Prior to birth the future dark cells reveal an increase in electron density after staining. At birth the morphological configuration of the dark cell area appeared quite mature.

The lipid composition and Ca transport function of sarcoplasmic reticulum(SR) membranes during development in vivo and in vitro.

Lipids play an essential role in the structure and function of biological membranes. The concept that the lipid enviroment influences the activity of membrane bound enzymes is now generally accepted. The sarcoplasmic reticulum(SR) of skeletal muscle is an intracellular membranous system with an important physiological role in contraction and relaxation (Hasselbach, 1964; Martonosi, 1971). The Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities which exhibit isolated SR preparations have been shown to be dependent on membrane phospholipids (Martonosi, 1968). An involvement of the fatty acid component of phospholipids in the functioning of the Ca2+ pump has also been suggested (Seiler et al., 1970; Seiler & Hasselbach, 1971). With the purpose of further exploring the relationships existing between lipids and other components of sarcoplasmic reticulum and its Ca transport function, changes in biochemical composition were correlated with variations in Ca2+ uptake during development of skeletal muscle in vivo and in vitro.