Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

Bmp4 and Noggin expression during early thymus and parathyroid organogenesis

The thymus and parathyroids originate from the third pharyngeal pouches, which form as endodermal outpocketings in the pharyngeal region beginning on embryonic day 9 (E9.0) of mouse development. Using organ-specific markers, we have previously shown that thymus and parathyroid-specific organ domains are established within the primordium prior to formation of the organs proper: Gcm2 expression defines the prospective parathyroid cells in the dorsal pouch from E9.5, while Foxn1 is expressed in the thymus domain from E11.25. Bmp (bone morphogenetic protein) signaling has been implicated in thymic epithelial cell differentiation and thymus organogenesis. In the present study, we report expression patterns of Bmp4 and Noggin, a Bmp4 antagonist, in the third pharyngeal pouch using two lacZ transgenic mouse strains. Results from this gene expression study revealed localization of Bmp4 expression to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5, in those cells that will express Foxn1 and form the thymus. Conversely, the expression of Noggin was confined to the dorsal region of the pouch and primordium at these stages, and thus appeared to be co-expressed with Gcm2 in the parathyroid domain. This represents the first detailed study of Bmp4 and Noggin expression during the early stages of thymus and parathyroid organogenesis.     

Developmental expression of plasma glutathione peroxidase during mouse organogenesis

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.     

Distinct enhancer elements control Hex expression during gastrulation and early organogenesis

In the mouse, embryological and genetic studies have indicated that two spatially distinct signalling centres, the anterior visceral endoderm and the node and its derivatives, are required for the correct patterning of the anterior neural ectoderm. The divergent homeobox gene Hex is expressed in the anterior visceral endoderm, in the node (transiently), and in the anterior definitive endoderm. Other sites of Hex expression include the liver and thyroid primordia and the endothelial cell precursors. We have used transgenic analysis to map the cis-acting regulatory elements controlling Hex expression during early mouse development. A 4.2-kb upstream region is important for Hex expression in the endothelial cell precursors, liver, and thyroid, and a 633-bp intronic fragment is both necessary and sufficient for Hex expression in the anterior visceral endoderm and the anterior definitive endoderm. These same regions drive expression in homologous structures in Xenopus laevis, indicating conservation of these regulatory regions in vertebrates. Analysis of the anterior visceral endoderm/anterior definitive endoderm enhancer identifies a repressor region that is required to downregulate Hex expression in the node once the anterior definitive endoderm has formed. This analysis also reveals that the initiation of Hex expression in the anterior visceral endoderm and axial mesendoderm requires common elements, but maintenance of expression is regulated independently in these tissues.     

Dynamic expression of manganese superoxide dismutase during mouse embryonic organogenesis

The balance between reactive oxygen species production and antioxidant defense enzymes in embryos is necessary for normal embryogenesis. To determine the dynamic expression profile of manganese superoxide dismutase (MnSOD) in embryos, which is an essential antioxidant enzyme in embryonic organogenesis, the expression level and distribution of MnSOD mRNA and protein were investigated in mouse embryos, as well as extraembryonic tissues on embryonic days (EDs) 7.5-18.5. MnSOD mRNA levels were remarkably high in extraembryonic tissues rather than in embryos during these periods. MnSOD protein levels were also higher in extraembryonic tissues than in embryos until ED 16.5, but the opposite trend was found after ED 17.5. MnSOD mRNA was observed in the chorion, allantois, amnion, ectoderm, ectoplacental cone and neural fold at ED 7.5 and in the neural fold, gut, ectoplacental cone, outer extraembryonic membranes and primitive heart at ED 8.5. After removing the extraembryonic tissues, the prominent expression of MnSOD mRNA in embryos was seen in the sensory organs, central nervous system and limbs on EDs 9.5-12.5 and in the ganglia, spinal cord, sensory organ epithelia, lung, blood cells and vessels, intestinal and skin epithelia, hepatocytes and thymus on EDs 13.5-18.5. Strong MnSOD immunoreactivity was observed in the choroid plexus, ganglia, myocardium, blood vessels, heapatocytes, pancreatic acinus, osteogenic tissues, brown adipose tissue, thymus and skin. These findings suggest that MnSOD is mainly produced from extraembryonic tissues and then may be utilized to protect the embryos against endogenous or exogenous oxidative stress during embryogenesis.     

Embryo developmental disruption during organogenesis produced by CF-1 murine periconceptional alcohol consumption

The aim was to study the control females (CF)-1 mouse embryo differentiation, growth, morphology on embryonic E- and N-cadherin expression at midgestation after periconceptional moderate alcohol ingestion. Adult female mice were exposed to 10% ethanol in drinking water for 17 days previous to and up to day 10 of gestation (ethanol-exposed females, EF) and were compared with nonexposed CF. EF presented reduced quantities of E10 to E10.5 embryos, greater percentage of embryos at stages less than E7.5, reduced implantation site numbers/female, and increased resorptions compared with CF. EF-embryo growth was significantly affected as evidenced by reduced cephalic and body sizes of E10 and E10.5 embryos (scanning electron microscopy) and decreased protein content of E10.5 embryos vs. CF embryos. A significantly higher percentage of EF-E10-10.5 embryos presented abnormal neural tube (NT) closure vs. the percentage of CF. E10 embryos from EF presented elevated tissue disorganization, pyknosis and nuclear condensation in somites, mesenchymal and neuroepithelial tissue. Immunohistochemical E- and N-cadherin distribution patterns were similar in organic structures of E10 embryos between groups. However, western blot revealed that E- and N-cadherin expression levels were significantly increased in EF-derived embryos vs. controls. Perigestational ethanol consumption by CF-1 mice induced significant damage in the organogenic embryogenesis by producing delayed differentiation, growth deficiencies, and increasing the frequency of NT defects. Ethanol exposure may disrupt cell-cell adhesion leading to upregulation of E- and N-cadherin expression suggesting that deregulation of cell adhesion molecules could be involved in the disruption of embryo development at organogenesis in CF-1 mouse.     

The BMP antagonist follistatin-like 1 is required for skeletal and lung organogenesis

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development.     

Changes in homing receptor expression on murine lymphokine-activated killer cells during IL-2 exposure

The effects of IL-2 on the expression of homing receptors by lymphocytes of NK or lymphokine activated killer (LAK) cell derivation has not yet been evaluated. We developed a murine model to evaluate the potential of LAK cells to localize into peripheral lymph nodes since LAK cells are used to treat human cancers which have metastasized to these tissues. Using a frozen section binding assay, LAK cell adhesion to the lymph node microvasculature was easily demonstrable. Inhibition studies demonstrated that LAK cell binding to lymph nodes was mediated by mechanisms previously described in T cells. LAK cell surface expression of the 85- to 95-kDa homing receptor recognized by the antibody MEL-14 on LAK cells was assessed by indirect immunofluorescence. The percentage of cells which bound MEL-14 decreased slightly over 3 days of IL-2 exposure (from 73 to 60%), particularly in the large granular lymphocyte (cytotoxic effector) subpopulation (45% MEL-14+). The expression of another homing-related molecule, leukocyte function-associated Ag-1, markedly increased during activation of LAK cells. Despite the expression of these homing receptors, we observed almost no LAK cell localization into lymph nodes in vivo. Furthermore, IL-2 pretreatment of recipient animals did not increase the adhesion of LAK cells to lymph node microvasculature or enhance their extravasation. IL-2 activation of non-T, non-B lymphocytes results in significant changes in both the expression and function of cell surface homing receptors. Our results indicate that in vitro analysis does not always predict in vivo localization potential.     

uPARAP expression during murine lung development

Lung remodeling requires active collagen deposition and degradation. Urokinase plasminogen activator receptor-associated protein (uPARAP), or Endo 180, is a cell-surface receptor for collagens, which leads to collagen internalization and degradation. Thus, uPARAP-mediated collagen degradation is an additional pathway for matrix remodeling in addition to matrix remodeling mediated by matrix metalloproteinases and cathepsins. Using immunohistochemistry, we demonstrate extensive uPARAP expression in the mesenchyme throughout murine lung development. By immunofluorescence, we demonstrate significant overlap of uPARAP expression with collagen IV expression, but minimal overlap with collagen I expression in the developing murine lung. Finally, we compared lung development between wild-type and uPARAP(-/-) mice, and found no significant histologic differences, indicating the presence of alternative collagen degradation pathways during murine lung development.     

Hexokinase I expression and activity in embryonic mouse heart during early and late organogenesis

 The role of Fas and Fas ligand (Fas-L) in the apoptotic cell death process in cisplatin (CP)-treated human proximal tubular epithelial cells (PTECs) was examined. The human PTECs were treated with various concentrations (20–80 μM) of CP for 24 h, and the incidence of apoptosis in CP-treated cells was assessed by trypan blue staining, propidium iodide staining, in situ end labeling, and electron microscopy. The expression of Fas and Fas-L was detected by immunofluorescence microscopy. The results showed that: (1) CP-treatment resulted in a decreased number of live human PTECs and an increased number of dead cells, (2) CP-treated human PTECs showed an increased rate of apoptosis with its typical morphological features, and (3) expression of both Fas and Fas-L was upregulated in CP-treated human PTECs. These results indicate that CP treatment induces apoptosis in human PTECs and the activation of the Fas/Fas-L system may play an active role in the induction of the apoptotic cell death process. Accepted: 13 January 1999