Directed differentiation of dendritic cells from mouse embryonic stem cells.

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.

The origin and efficient derivation of embryonic stem cells in the mouse.

By explanting tissues isolated microsurgically from implanting strain 129 mouse blastocysts individually on STO feeder cells we have established that embryonic stem (ES) cells originate from the epiblast (primitive ectoderm). Isolated early epiblasts yielded ES cell lines at a substantially higher frequency than intact blastocysts regardless of whether they were explanted whole or as strictly single-cell suspensions. When explanted from delayed-implanting 129 blastocysts, epiblasts gave lines consistently in 100% of cases. If primary embryonic fibroblasts rather than STO cells were used as feeders, germline-competent ES cell lines were obtained readily from epiblasts of delayed-implanting blastocysts of several hitherto refractory strains, particularly when recombinant leukemia inhibitory factor was included in the medium during the initial period of culture. Because lines were obtained from the nonpermissive CBA/Ca strain at a rate of up to 56%, this approach to the derivation of germline-competent ES cell lines may not only prove generic for the mouse but also worth pursuing in other species of mammal.

Reflections on the biology of embryonic stem (ES) cells.

Remarkably little is known about mammalian embryonic stem (ES) cells despite their very widespread use in studies on gene disruption and transgenesis. As yet, it is only in the mouse that lines of ES cells which retain the ability to form gametes following reintroduction into the early conceptus have been obtained. Even in this species, most strains have so far proved refractory to the derivation of such cell lines. Apart from persisting ignorance as to how the various procedures that have been claimed to improve success actually do so, even the tissue of origin of ES remains uncertain. Furthermore, it is doubtful whether retention of pluripotency or expression of so-called "stem cell" marker molecules provide an adequate basis for classifying cells as genuine ES cells. This is because epiblast cells, their presumed precursors, lose the capacity to colonize the preimplantation conceptus well before they become restricted in the types of cell they can form or cease to express such marker molecules. In addition, it has yet to be established whether heterogeneity of cells within individual ES cell lines arises entirely during culture or is at least partly attributable to lack of homogeneity among their precursors. Finally, it has yet to be explained why ES chimeras evidently differ from those obtained by combining cells from different conceptuses in showing greater variation between tissues in the level of chimerism.

Female predisposition to cranial neural tube defects is not because of a difference between the sexes in the rate of embryonic growth or development during neurulation.

The susceptibility of females to anencephaly is well established and has been suggested to result from a slower rate of growth and development of female embryos during cranial neurulation. We have tested this hypothesis by measuring the rates of growth and development, both in utero and in vitro, of male and female embryos of the curly tail (ct) mutant mouse strain, in which cranial neural tube defects occur primarily in females. Embryonic growth was assessed by increase in protein content, while development progression was judged from increase in somite number and morphological score. Embryos were sexed by use of the polymerase chain reaction to amplify a DNA sequence specific to the Y chromosome, and by sex chromatin analysis. We find that, during neurulation (between 8.5 and 10.5 days of gestation), males are advanced in growth and development relative to their female litter mates, but that the rates of growth and development do not differ between the sexes during this period. We conclude that rate of embryonic growth and development is unlikely to determine susceptibility to cranial neural tube defects. It seems more likely that male and female embryos differ in some specific aspect(s) of the neurulation process that increases the susceptibility of females to development of anencephaly.

Interaction between splotch (Sp) and curly tail (ct) mouse mutants in the embryonic development of neural tube defects.

The mouse mutations splotch (Sp) and curly tail (ct) both produce spinal neural tube defects with closely similar morphology, but achieve this by different embryonic mechanisms. To determine whether the mutants may interact during development, we constructed mice carrying both mutations. Double heterozygotes exhibited tail defects in 10% of cases, although the single heterozygotes do not express this phenotype. Backcrosses of double heterozygotes to ct/ct produced offspring with an elevated incidence of neural tube defects, both spina bifida and tail defects, compared with a control backcross in which Sp was not involved. Use of the deletion allele Sp2H permitted embryos carrying a splotch mutation to be recognised by polymerase chain reaction assay. This experiment showed that only embryos carrying Sp2H develop spina bifida in the backcross with ct/ct, suggesting that the genotype Sp2H/+, ct/ct is usually lethal around the time of birth as a result of severe disturbance of neurulation. The interaction between Sp and ct was investigated further by examining embryos in the backcross for developmental markers of the Sp/Sp and ct/ct genotypes. Sp/Sp embryos characteristically lack neural crest derivatives, such as dorsal root ganglia, and die on day 13 of gestation. Double mutant embryos from the backcross did not exhibit either of these characteristics suggesting that homozygosity for ct does not cause Sp/+ embryos to develop as if they were of genotype Sp/Sp. The angle of ventral curvature of the posterior neuropore region is enhanced in affected ct/ct embryos whereas it was found to be reduced in Sp/Sp embryos compared with their normal littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of neural tube defects in a mouse mutant using whole embryo culture.

The method of whole embryo culture permits a variety of experimental manipulations to be performed on the mammalian embryo. When used in conjunction with mouse mutants, this technique can provide information on the pathogenetic mechanisms underlying the development of birth defects. To illustrate this approach, we review in vitro studies on the development of embryos homozygous for the mutation curly tail (ct). These studies have involved making repeated observations on individual embryos, performing surgical manipulations, applying environmental influences and metabolic labelling. As a result of this work, we have now partially elucidated the developmental sequence of events that precedes the appearance of spina bifida in the ct mutant.

Exogenous transferrin is taken up and localized by the neurulation-stage mouse embryo in vitro.

We have screened neurulation-stage mouse embryos for regional differences in protein distribution, by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The screen has revealed an 83-kD protein (pI 6.8) that is present in embryo regions where neurulation is in progress but not in regions where neurulation is complete. The 83-kD protein is not synthesized in the neurulation-stage embryo or in the yolk sac, but is taken up from the culture serum in vitro and, probably, from the maternal serum in utero. The 83-kD protein has been identified as transferrin on the basis of its electrophoretic migration and recognition on Western blots by an antitransferrin antibody. Culture of embryos in serum containing 125I-transferrin, followed by autoradiography of embryo sections, shows that transferrin is taken up and localized in the gut beneath the closing neural folds at several levels of the body axis in 8.5- and 9.5-day embryos. In situ hybridization studies show that the transferrin receptor mRNA is expressed in all cells of the 9.5-day embryo, including the gut endoderm. These findings are consistent with a role for transferrin in development of the gut and perhaps, indirectly, in completion of neurulation during early mouse embryogenesis.

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.

Curvature of the caudal region is responsible for failure of neural tube closure in the curly tail (ct) mouse embryo.

Delayed closure of the posterior neuropore (PNP) occurs to a variable extent in homozygous mutant curly tail (ct) mouse embryos, and results in the development of spinal neural tube defects (NTD) in 60% of embryos. Previous studies have suggested that curvature of the body axis may delay neural tube closure in the cranial region of the mouse embryo. In order to investigate the relationship between curvature and delayed PNP closure, we measured the extent of ventral curvature of the neuropore region in ct/ct embryos with normal or delayed PNP closure. The results show significantly greater curvature in ct/ct embryos with delayed PNP closure in vivo than in their normal littermates. Reopening of the posterior neuropore in non-mutant mouse embryos, to delay neuropore closure experimentally, did not increase ventral curvature, suggesting that increased curvature in ct/ct embryos is not likely to be a secondary effect of delayed PNP closure. Experimental prevention of ventral curvature in ct/ct embryos, brought about by implantation of an eyelash tip longitudinally into the hindgut lumen, ameliorated the delay in PNP closure. We propose, therefore, that increased ventral curvature of the neuropore region of ct/ct embryos imposes a mechanical stress, which opposes neurulation and thus delays closure of the PNP. Increased ventral curvature may arise as a result of a cell proliferation imbalance, which we demonstrated previously in affected ct/ct embryos.

Does lumbosacral spina bifida arise by failure of neural folding or by defective canalisation?

The aim of this study was to determine whether open lumbosacral spina bifida results from an abnormality of neural folding (primary neurulation) or medullary cord canalisation (secondary neurulation). Homozygous curly tail (ct) mouse embryos were studied as a model system for human neural tube defects. The rostral end of the spina bifida was found to lie at the level of somites 27 to 32 in over 90% of affected ct/ct embryos. Indian ink marking experiments using non-mutant embryos showed that the posterior neuropore closes, and primary neurulation is completed, at the level of somites 32 to 34. Since neurulation in mammals progresses in a craniocaudal sequence, without overlap between regions of primary and secondary neurulation, we conclude that spina bifida in ct/ct embryos arises initially as a defect of primary neurulation. The position of posterior neuropore closure in human embryos is estimated to lie at the level of the future second sacral segment indicating that in humans, as in the ct mouse, lumbosacral spina bifida usually arises as a defect of posterior neuropore closure. Cranial NTD affect females predominantly, whereas lower spinal NTD are more common in males, both in humans and ct mice. We offer an explanation for this phenomenon based on (a) differences in the effect of embryonic growth retardation on the likelihood that an embryo will develop either cranial or lower spinal NTD and (b) differences in the rate of growth and development of male and female embryos at the time of neurulation.

Ovarian interstitial tissue of the wood mouse, Apodemus sylvaticus.

The ovary of the wood mouse contains an unusually large amount of interstitial tissue which appears to develop from undifferentiated cells of the ovarian stroma and also by transformation of theca or granulosa cells of atretic follicles. The cells are characterized by the presence of smooth endoplasmic reticulum, rounded mitochondria with tubular cristae, and abundant, large (1.5 micron diameter) lipid droplets containing cholesterol and its esters. 3 beta-Hydroxysteroid dehydrogenase activity occurs in the interstitial cells. Their ultrastructural characteristics suggest that the cells are not very active, but their abundance and the considerable amount of steroid hormone precursor they contain may compensate for low secretory activity and they may be an important and (from a developmental viewpoint) early source of steroid hormone.

A cell-type-specific abnormality of cell proliferation in mutant (curly tail) mouse embryos developing spinal neural tube defects.

The mouse mutant curly tail (ct) provides a model system for studies of neurulation mechanisms. 60% of ct/ct embryos develop spinal neural tube defects (NTD) as a result of delayed neurulation at the posterior neuropore whereas the remaining 40% of embryos develop normally. In order to investigate the role of cell proliferation during mouse neurulation, cell cycle parameters were studied in curly tail embryos developing spinal NTD and in their normally developing litter-mates. Measurements were made of mitotic index, median length of S-phase and percent reduction of labelling index during a [3H]thymidine pulse-chase experiment. These independent measures of cell proliferation rate indicate a reduced rate of proliferation of gut endoderm and notochord cells in the neuropore region of embryos developing spinal NTD compared with normally developing controls. The incidence of cell death and the relative frequency of mitotic spindle orientations does not differ consistently between normal and abnormal embryos. These results suggest a mechanism of spinal NTD pathogenesis in curly tail embryos based on failure of normal cell proliferation in gut endoderm and notochord.

Prevention of spinal neural tube defects in the mouse embryo by growth retardation during neurulation.

Homozygous mutant curly tail mouse embryos developing spinal neural tube defects (NTD) exhibit a cell-type-specific abnormality of cell proliferation that affects the gut endoderm and notochord but not the neuroepithelium. We suggested that spinal NTD in these embryos may result from the imbalance of cell proliferation rates between affected and unaffected cell types. In order to test this hypothesis, curly tail embryos were subjected to influences that retard growth in vivo and in vitro. The expectation was that growth of unaffected rapidly growing cell types would be reduced to a greater extent than affected slowly growing cell types, thus counteracting the genetically determined imbalance of cell proliferation rates and leading to normalization of spinal neurulation. Food deprivation of pregnant females for 48 h prior to the stage of posterior neuropore closure reduced the overall incidence of spinal NTD and almost completely prevented open spina bifida, the most severe form of spinal NTD in curly tail mice. Analysis of embryos earlier in gestation showed that growth retardation acts by reducing the incidence of delayed neuropore closure. Culture of embryos at 40.5 degrees C for 15-23 h from day 10 of gestation, like food deprivation in vivo, also produced growth retardation and led to normalization of posterior neuropore closure. Labelling of embryos in vitro with [3H]thymidine for 1 h at the end of the culture period showed that the labelling index is reduced to a greater extent in the neuroepithelium than in other cell types in growth-retarded embryos compared with controls cultured at 38 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)