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A mouse mammary tumor, adenocarcinoma BW 10232, was maintained in vitro for 14 days, separated from embryonic mammary mesenchyme by a Millipore filter. Tubules developed in the tumor; deoxyibonucleic acid synthisis declined; and a presumptive acid mucopolysaccharide matrix, not evident in the controls, appeared.
Although the development of the inner ear has been a favorite subject for biologists to study, it is not yet clear exactly which molecules are involved in the induction of the otic placode, the plug of embryonic ectoderm that will become the inner ear. In his Perspective, Graham takes us on an inner ear odyssey, explaining how the signaling molecules FGF-19 and Wnt-8c cooperate to induce formation of the otic placode (Ladher et al.).
Embryonic stem cells represent an attractive source of cardiomyocytes for cell-replacement therapies. However, before embryonic stem cells can be successfully used for the treatment of cardiac diseases, the precise molecular mechanisms that underlie their cardiogenic differentiation must be identified. A network of intrinsic and extrinsic factors regulates embryonic stem cell self-renewal and differentiation into a variety of different cell lineages. Here, we show that Notch signaling takes place in some but not all embryonic stem cells and that the Notch pathway is shut down during the course of differentiation concomitantly with downregulation of Notch receptor and ligand expression. Moreover, gain- and loss-of-function experiments for Notch signaling components show that this pathway is a crucial regulator of cardiomyocyte differentiation within ES cells. Differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch1 receptor, whereas endogenous Notch signaling promotes differentiation of ES cells into the neuronal lineage. We conclude that Notch signaling influences the cell fate decision between mesodermal and the neuroectodermal cell fates during embryonic stem cell differentiation. These findings should help to optimize the production of specific cell types via modulation of the Notch pathways and, in particular, to improve the production of embryonic stem cell-derived cardiomyocytes.
Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/betageo/phosphoglycerate kinase-hygromycin-resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14-day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day-16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X-gal staining after neomycin selection, (b) BrdU (bro-modeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C-peptide, and glucose transporter 2 (Glut-2), (d) reverse transcription-polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX-1), (e) insulin and C-peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch-clamp studies in inside-out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin-diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX-1; coexpression of insulin, C-peptide, and Glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering beta cells from stem cells.
Embryonic stem (ES) cells are pluripotential cells, and enable us to study mechanisms of cell differentiation. Gene disruption of ES cells by homologous recombination is to be clear the function of targeted genes. Recently, it has been reported that bone marrow hematopoietic stem cells have a potential to differentiate into neuronal cell, muscle cell, liver cell, epidermal cell, and also epithelial cell lineages. Moreover, cloned animals from somatic cell nuclei were produced. Here, we show osteoclastogenesis, and endothelial cell-genesis from single ES cell, and discuss the possibility for organogenesis in vitro. Furthermore, we would like to summon to understand usefulness and dangerousness of the regenerative medicine.
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The mRNAs for 2 isotypes of alpha-tubulin, termed T alpha 1 and T26, are differentially regulated in the developing rat nervous system. T alpha 1 alpha-tubulin mRNA is expressed at high levels when neurons extend processes whereas T26 mRNA is expressed constitutively (Miller et al., 1987b). We have examined the expression of these 2 alpha-tubulin mRNAs in regenerating facial and sciatic motor neurons of the rat using Northern blot and in situ hybridization analyses. T alpha 1 alpha-tubulin mRNA is rapidly induced in axotomized motor neurons of the facial nerve: increased levels of mRNA are detectable 4 hr after a lesion is made 1.5 cm distal to the neuronal cell bodies. T alpha 1 mRNA levels are highest from 3-7 d postcrush and decline slowly to control levels following functional reinnervation of facial muscles. In contrast, T26 mRNA levels remain constant throughout the regeneration process. Total alpha-tubulin mRNA levels do not change until 1 d postaxotomy; otherwise the changes in expression are similar to T alpha 1 mRNA, although the relative increase is not as great. Enhanced T alpha 1 alpha-tubulin mRNA expression also occurs in motor neurons of crushed or tied sciatic nerve. Ligature or crush of the sciatic nerve leads to approximately the same peak in the expression of T alpha 1 mRNA at 7-15 d postaxotomy. Following the facial nerve transection, under conditions in which reinnervation is prevented, T alpha 1 alpha-tubulin mRNA levels remain elevated significantly longer than when the nerve is crushed. Taken together, the data indicate that T alpha 1 alpha-tubulin mRNA is rapidly induced following neuronal axotomy, remains elevated during the period of axonal regrowth, and is subsequently down-regulated at the approximate time of target contact. These results are reminiscent of changes in T alpha 1 mRNA that occur during neuronal development. This growth-associated pattern of T alpha 1 gene expression can be modified by inhibiting appropriate regeneration of the damaged nerve.
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The biological activity of 26-kD membrane-bound tumor necrosis factor (TNF)-a in embryonal development was examined in an in vitro system using embryonic stem (ES) cells. ES cells were seeded on NIH3T3 feeder cells transformed with mouse precursor TNF-a gene to express membrane-bound TNF-a on their cell surface. The proliferation of the ES cells was reduced and differentiation was accelerated. The same effects were also observed when 17-kD free TNF-a was added to the culture medium of the ES cells. Since free TNF-a is not present during embryogenesis, these results suggest that membrane-bound TNF-a may play an important role in embryonal development through cell-cell contact.
The effects of several chelators, including hinokitiol, on embryonal carcinoma F9 cell differentiation were assessed by assaying the production of plasminogen activator (PA) as a differentiation marker protein. Hinokitiol and tropolone which were potent differentiation inducers lost their activity following preincubation with Fe2+ and Fe3+ ions. Other metal ions had no or little effect on the hinokitiol-induced differentiation. Of several chelators examined, dithizone induced differentiation as effectively as did hinokitiol and tropolone. Dithizone-induced differentiation was also inhibited by preincubation with Fe3+ ions. It was concluded that some potent iron chelators could trigger the teratocarcinoma F9 cells to differentiate through the chelation with intracellular iron ions.
OBJECTIVE: To investigate the effect of anti-fecundity and anti-embryonation immunity on liver granuloma formation in mice immunized with soluble immature egg antigen (SIEA) of schistosoma japonicum. METHOD: BALB/c mice were immunized with the crude SIEA or 26,000-28,000 molecular antigen derived from SIEA and compared the size in schistosoma japonicum egg granulomas in the liver after challenge infection both the immunized mice and unimmunized controls. RESULTS: The number and size of egg granuloma were significantly decreased in mice immunized with SIEA and SIEA 26-28,000, by 29.26% and 49.64%, respectively. The inhibitive effects on granuloma formation and spleen weights in the group immunized with SIEA 26-28,000 were stronger than those in the group immunized with the crude SIEA. However, mean granuloma size and spleen weights in the group immunized with SEA or SIEA-1 fraction were not different from unimmunized control. CONCLUSION: SIEA and SIEA 26-28,000 antigenic molecule play an important role in inhibition on liver granuloma formation through their anti-embryonation and anti-fecundity immunity effects.
Analysis of two-dimensional gel electrophoretic patterns of proteins labeled with [35S]methionine was undertaken to investigate changes in gene expression in embryonal carcinoma cell line Nulli-SCC1 during the induction of differentiation in vitro by retinoic acid. The results indicate that changes in protein profiles occur long before overt morphological differentiation is observed. Studies utilizing the antimetabolite, alpha-amanitin, coupled with those involving the cell-free translation of poly(A)+ -RNA, suggest that gene expression is controlled post-transcriptionally as well as transcriptionally. In this respect, embryonal carcinoma cells appear to resemble early embryonic cells, which show evidence of multiple levels of control of gene expression during the initial stages of development and differentiation.
PCC4azal embryonal carcinoma tumors were grown in strain 129 mice by s.c. transplantation. When palpable, the tumors were treated with a combination of retinoic acid and dimethylacetamide. In vitro, this embryonal carcinoma cell line shows minimal spontaneous differentiation and is exquisitely sensitive to retinoic acid and/or dimethylacetamide induction of differentiation. Ten daily 20-microliter intratumor injections of a solution of 10 mg retinoic acid per ml of dimethylacetamide resulted in nearly complete induction of morphological differentiation mainly into neuroepithelial and glandular derivatives. Control tumors showed minor spontaneous differentiation. Differentiation was associated with decreased tumor growth rate, decreased mitotic index, decreased extent of necrosis, and increased survival time of the hosts. In 4 of 18 cases, long-term survival of the hosts was effected by a complete differentiation of the malignant embryonal carcinoma tumors into benign teratomas. Retinoic acid:dimethylacetamide was also effective in inducing differentiation with the same dosage and schedule when administered systemically, i.e., i.p. or s.c.
Few molecular comparisons have been made between the processes of embryogenesis and regeneration or transdifferentiation that lead to the formation of the same structures. In the amphibian, Xenopus laevis, the cornea can undergo transdifferentiation to form a lens when the original lens is removed during tadpole larval stages. Unlike the process of embryonic lens induction, cornea-lens transdifferentiation is elicited via a single inductive interaction involving factors produced by the neural retina. In this study, we compared the expression of a number of genes known to be activated during various phases of embryonic lens formation, during the process of cornea-lens transdifferentiation. mRNA expression was monitored via in situ hybridization using digoxigenin-labeled riboprobes of pax-6, Xotx2, xSOX3, XProx1, and gamma6-cry. We found that all of the genes studied are expressed during both embryogenesis and cornea-lens transdifferentiation, though in some cases their relative temporal sequences are not maintained. The reiterated expression of these genes suggests that a large suite of genes activated during embryonic lens formation are also involved in cornea-lens transdifferentiation. Ultimately functional tests will be required to determine whether they actually play similar roles in these processes. It is significant that the single inductive event responsible for initiating cornea-lens transdifferentiation triggers the expression of genes activated during both the early and late phases of embryonic lens induction. These findings have significant implications in terms of our current understanding of the "multistep" process of lens induction. Dev Dyn 1999;215:308-318.
We have developed a novel induction gene trap approach that preselects in vitro for integrations into genes that lie downstream of receptor/ligand-mediated signaling pathways. Using this approach, we have identified 20 gene trap integrations in embryonic stem cells, 9 of which were induced and 11 of which were repressed after exposure to exogenous retinoic acid (RA). All but one of these integrations showed unique spatially restricted or tissue-specific patterns of expression between 8.5 and 11.5 days of embryogenesis. Interestingly, expression was observed in tissues that are affected by alterations in RA levels during embryogenesis. Sequence analysis of fusion transcripts from six integrations revealed five novel gene sequences and the previously identified protooncogene c-fyn. To date, germ-line transmission and breeding has uncovered one homozygous embryonic lethal and three homozygous viable insertions. These studies demonstrate the potential of this induction gene trap approach for identifying and mutating genes downstream of signal transduction pathways.
The induction of a frequent fusion in dissociated fish embryonic cells is reported. Fusion was induced during mechanical isolation of blastomeres in normal saline solution or Ca2+-free saline solution by quickly bringing about physical contact between cells within about 1-5 min of dissociation. Fused blastomeres were obtained in about 18% yield from early morula cells in about 68% yield from early blastula cells and in about 5% yield from early gastrula cells.