Embryonic development of the mouse mutant pupoid foetus (pf/pf)

Summary The pupoid foetus mutation in the mouse is a recessive lethal mutation causing death of homozygous (pf/pf) embryos immediately after birth. From 11.3 days gestation onwards, these embryos are characterised externally by the development of a tail twist, followed by apparent stunting of the limbs and tail (when compared with the development of these structures in normal embryos), lack of digits, distortion of facial features, and possession of a smooth, mottled skin. Embryos ranging in age from 11.3 days gestation to full term have been examined using light microscopy and scanning and transmission electron microscopy. The skeletal structure and internal organs of the embryo are normal, but abnormalities occur in the external epidermis, the dermis, and the peripheral sensory nerves. Development of the palate and the eyes are affected by the behaviour of these tissues.The epidermis undergoes hypertrophy and fails to differentiate, and, on the basis of morphological criteria and theoretical considerations, it is suggested that the pf gene is activated in the epidermis during the keratinization pathway, preventing differentiation and altering the cell surface characteristics of the cells. Other abnormalities are explained in terms of interactions with the epidermis. This mutant is compared with other similar mutants.     

Eye development in the normal and Pupoid foetus (pf/pf) mutant mouse

Summary Embryonic development of the mammalian eye has not been studied in such great detail as that of the avian eye, and preliminary observations have suggested that the sequence of events may differ. It is therefore likely that the relative importance of the cell and tissue interactions involved also differs and it would be interesting to compare these two systems.The pupoid foetus mutation in the mouse shows disruption of eye development due to abnormal epidermal properties and so the relative importance of individual events in triggering subsequent development can be studied by seeing what happens when the situation is modified in the mutant.The behaviour of the pupoid foetus epidermal cells in the interactive system of the eye may also help to further characterise the phenotype of the mutation.     

The organ distribution of gp-330 (Heymann antigen) and gp-90 in the mouse and the rat

Summary The Heymann antigen (gp-330) and an antigen with lower molecular weight (gp-90) are major constituents of the brush border of the renal proximal tubules in the rat and the mouse. The Heymann antigen can also be found at discrete sites in the glomerular visceral epithelium of the rat, but not of the mouse. Gp-90 is present diffusely along the glomerular capillary wall of rat and mouse. The Heymann antigen is probably the target antigen for membranous glomerulonephritis in the rat, while in the mouse, where this form of glomerulonephritis can also be induced, gp-90 seems to be the antigen involved. We have separated the antibody populations against these two antigens by preparing eluates from kidneys of rats and livers of mice that had been injected with an antiserum against pronase-digested mouse renal tubular antigens. Using these purified antibodies we have examined by indirect immunofluorescence the distribution of the two antigens on normal mouse and rat tissues. The expression of the Heymann antigen is limited to the epithelia of several organs, while gp-90 has a more widespread distribution in many cells of different origin and function in both the mouse and the rat.     

Histopathology of the late-onset motor neuron degeneration (Mnd) mutant in the mouse

The motor neuron degeneration (Mnd) is characterized by a progressive deterioration of motor function (stiff-legged gait, abnormal limb placements and grasping, and finally paralysis; moving from rear to forelimbs). There is a dramatic degeneration of spinal cord motor neurons, more severe in the lumbosacral than in the other regions, as well as variable pathology in the lower cranial nerves. Upper motor neurons of the red nucleus, reticular formation of the pons and medulla, and restricted areas of the cerebral cortex are also affected. Degenerating motor neurons share many characteristics seen in the human disease amyotropic lateral sclerosis, including loss of Nissl substance, increases in lipofuscin and abnormal cytoplasmic inclusions. Additionally, Mnd, like ALS, is a disease of later life.     

Histopathology of the late-onset motor neuron degeneration (Mnd) mutant in the mouse

The motor neuron degeneration (Mnd) is characterized by a progressive deterioration of motor function (stiff-legged gait, abnormal limb placements and grasping, and finally paralysis; moving from rear to forelimbs). There is a dramatic degeneration of spinal cord motor neurons, more severe in the lumbosacral than in the other regions, as well as variable pathology in the lower cranial nerves. Upper motor neurons of the red nucleus, reticular formation of the pons and medulla, and restricted areas of the cerebral cortex are also affected. Degenerating motor neurons share many characteristics seen in the human disease amyotropic lateral sclerosis, including loss of Nissl substance, increases in lipofuscin and abnormal cytoplasmic inclusions. Additionally, Mnd, like ALS, is a disease of later life.     

Induction and prevention of carcinogen-induced precancerous lesions in mouse mammary gland organ culture

Mouse mammary glands respond to growth promoting hormones in organ culture. In the presence of insulin, prolactin, aldostrone, and hydrocortisone, the glands exhibit extensive proliferation within 10 days of culture mimicking the mammary alveolar structures observed during pregnancy. However withdrawal of prolactin and steroids from the medium for an additional 14 days results in the disintegration of the alveolar structures resembling the mammary morphology observed during the involution stage. During the growth promoting phase if the glands are exposed to 7, 12, dimethylbenz(a)anthracene (DMBA) for 24 hours and cultured through the entire 24 days of culture period, they develop precancerous lesions. This model is highly reproducible and extensively utilized to evaluate efficacy of potential chemopreventive agents against carcinogen-induced mammary lesions.     

An improved method for culture of epidermal keratinocytes from newborn mouse skin

Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture medium containing low Ca²⁺ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin v6. The expression level of v6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.     

A simple organ culture method for differentiation of melanocytes in mouse embryonic skin using “origami” membrane filter

Summary A simple organ culture method for culturing embryonic skin was developed. A piece of skin with a part of the neural tube from mouse embryo (11 to 12 d) was placed on a 25 mm d membrane filter. The filter was folded to wrap the explant and inserted into glass tubing. The explant and filter in the glass tubing were placed in a rotating tissue culture tube containing 5 ml culture medium (Ham's F12 supplemented with 15 to 20% fetal bovine serum) and filled with a mixture of 95% air:5% CO₂. In explants cultured for 6 d fully differentiated melanocytes were observed in the epidermis.     

Relationship between altered axial curvature and neural tube closure in normal and mutant (curly tail) mouse embryos

Neural tube defects, including spina bifida, develop in the curly tail mutant mouse as a result of delayed closure of the posterior neuropore at 10.5 days of gestation. Affected embryos are characterized by increased ventral curvature of the caudal region. To determine whether closure of the neuropore could be affected by this angle of curvature, we experimentally enhanced the curvature of non-mutant embryos. The amnion was opened in 9.5 day embryos; after 20 h of culture, a proportion of the embryos exhibited a tightly wrapped amnion with enhanced curvature of the caudal region compared with the control embryos in which the opened amnion remained inflated. Enhanced curvature correlated with a higher frequency of embryos with an open posterior neuropore, irrespective of developmental stage within the range, 27–32 somites. Thus, within this somite range, caudal curvature is a more accurate determinant for normal spinal neurulation than the exact somite stage. Enhanced ventral curvature of the curly tail embryo correlates with an abnormal growth difference between the neuroepithelium and ventral structures (the notochord and hindgut). We experimentally corrected this imbalance by culturing under conditions of mild hyperthermia and subsequently determined whether the angle of curvature would also be corrected. The mean angle of curvature and length of the posterior neuropore were both reduced in embryos cultured at 40.5°C by comparison with control embryos cultured at 38°C. We conclude that the sequence of morphogenetic events leading to spinal neural tube defects in curly tail embryos involves an imbalance of growth rates, which leads to enhanced ventral curvature that, in turn, leads to delayed closure of the posterior neuropore.     

Integration of a gene marker into mouse mammary glands during whole organ culture

The objective of this study was to develop a method to indelibly mark mammary cells to follow cell lineage. This was done by infecting mouse mammary glands in whole organ culture (WOC) with a retroviral vector containing the -galactosidase (BAG) gene. Abdominal mammary glands from BALB/c mice primed with estrogen and progesterone were removed and injected with 30 µl of concentrated vector prepared from CRE BAG 2 (ATCC 1858 — CRL) cells or left uninjected and co-cultured with CRE BAG 2 feeder layers for 48 or 96 hours. Glands were floated on siliconized lens paper and incubated in Waymouth's 752/1 medium supplemented with insulin, aldosterone, hydrocortisone, prolactin, and epidermal growth factor for 5 days. Integration of BAG was determined with a colorimetric assay using X-gal, a -galactoside analog, in whole mounts and collagenase digested glands. Blue cells in collagenase digests and blue areas in whole mounts indicated that BAG had integrated into mammary cells during whole organ culture.Abbreviations A aldosterone - BAG -galactosidase - EGF epidermal growth factor - E estrogen - FCS fetal calf serum - H hydrocortisone - I insulin - Prl prolactin - P progesterone - WOC whole organ culture     

Cell kinetics of human gastric mucosa and gastric cancer in organ culture

Summary The cell kinetics of human gastric epithelium in organ culture have been measured using flash labelling with tritiated thymidine and the metaphase arrest technique to estimate cell birth rates. Normal gastric antral and body mucosa have been compared with mucosa showing gastritis and gastric carcinoma. Labelling indices with tritiated thymidine in normal gastric mucosa declined over a 48-h period suggesting that essential growth factors were lacking. Labelling indices and cell birth rates were higher in gastritis than in normal mucosa and highest in gastric carcinoma. Labelling indices were higher in intestinal-type gastric carcinoma than diffuse carcinoma. In metaphase arrest experiments carcinomas showed on average an eightfold increase in resistance to the metaphase-arresting properties of vincristine when compared with normal mucosa. The validity of using the metaphase arrest technique to measure cell birth rate in gastric cancers in view of this vincristine resistance is discussed.     

Epithelial-mesenchymal interactions in differentiation of stomach epithelium in fetal mice

Summary Epithelial-mesenchymal interactions during development of the forestomach and glandular stomach in fetal mice were investigated by recombination experiments in vitro. Stomach epithelium could not survive when cultivated alone, but its development was supported by the presence of homologous or heterologous mesenchyme.The developmental fate of the epithelium was not affected by recombination with heterologous mesenchyme, but the expression of epithelial differentiation was influenced by the type of mesenchyme. The rate of keratinization of the forestomach epithelium was significantly greater on recombination with homologous mesenchyme than on recombination with heterologous mesenchyme. Moreover, the rate of formation of glandular structures in the glandular stomach epithelium was significantly greater on recombination with 16.5-day stomach mesenchyme than on recombination with 14.5- or 18.5-day stomach mesenchyme.     

From skin dendritic cells to a simplified classification of human and mouse dendritic cell subsets

Recent studies have identified several DC subsets within the mouse skin and showed that functional specialization exists among them. This Viewpoint summarizes recent data on functional specialization of skin DC subsets and integrates this knowledge into a unifying DC classification that emphasizes the similarities between the DC subsets found in both lymphoid and nonlymphoid tissues of several mammalian species.     

Antibody-mediated lysis of the bovine subcommissural organ maintained in culture

The subcommissural organ (SCO) is a brain gland that secretes glycoproteins into the cerebrospinal fluid (CSF). It is an ancient and conserved secretory structure of the brain, developing very early in ontogeny. However, the function of the SCO is unknown. The secretory cells of the SCO are arranged into a single or double, irregularly shaped layer located at the interface of the CSF and nervous tissue. This has prevented its selective surgical destruction. The present investigation was designed to destroy the secretory cells of 30-day-old expiants of bovine SCO by use of an immunological approach. A membrane preparation enriched with plasma membrane of the secretory cells of the bovine SCO was obtained. This preparation was further processed to separate the structural proteins. A similar procedure was applied to obtain a fraction of integral proteins of the plasma membrane of a nonsecretory ciliated ependyma. Antisera were prepared against both preparations of integral proteins. The antiserum against the fraction obtained from the SCO cells immunostained the plasma membrane of the bovine SCO cells and in immunoblot it reacted with several proteins of the membrane preparation from SCO cells. When added to the culture medium this antibody bound to the apical plasma membrane of the secretory ependyma of the bovine SCO kept in culture; it caused the lysis of these cells when used together with complement. None of these properties were displayed by the antiserum raised against the integral proteins of the plasma membrane of the ciliated ependyma. This antiserum, however, immunostained the bovine ciliated ependyma neighboring the SCO. These results indicate that immunological surgery of the SCO in living animals may be possible to achieve.     

不同超排卵方案和小鼠品系来源的2-细胞期胚胎体外培养比较

目的比较激素注射间隔时间差异对昆明系小白鼠（KM小鼠）超数排卵的影响以及KM小鼠和ICR小鼠2-细胞胚胎的体外发育能力。方法29只KM小鼠随机分成两组,分别间隔48h和72h注射10IU的孕马血清（PMSG）和尿绒毛膜促性腺激素（HCG）,比较获取2-细胞胚胎数以及其体外发育能力的差异;分7次同时常规超排KM小鼠和ICR小鼠,体外培养2-细胞胚胎,记录囊胚形成数。结果48h超排卵组获取2-细胞期小鼠胚胎数目显著多于72h超排卵组（P〈0．01）,但前者的囊胚形成率却显著低于后者,两者间差异有统计学意义（P〈0．05）。KM小鼠和ICR小鼠2-细胞胚胎的囊胚形成率间无显著差异（P〉0．05）。结论间隔48h超排KM小鼠可获取较多胚胎,但间隔72h超排所获2-细胞期小鼠胚胎体外发育潜能好;小鼠品系不影响2-细胞胚胎的体外培养结果。     

小鼠卵母细胞体外成熟培养系统的建立与优化

目的 探讨激素刺激时间、体外培养时间对小鼠卵母细胞核成熟及不同激活方案对卵母细胞孤雌激活、胚胎发育能力的影响。方法 孕马血清促性腺激素（PMSG）超排处理,在体外培养的不同时间点检测卵母细胞核成熟率（每个处理至少重复3次,3只/重复,以下实验相同）。
分别采用乙醇结合6-二甲氨基嘌呤（6-DMAP）法和SrCl₂ 法激活卵母细胞,胚胎培养液选用CZB［胎牛血清（FBS）或牛血清清蛋白（BSA）］两种,确定最佳激活方案。对不同时间点成熟的卵母细胞进行激活,确定最佳激活卵龄。研究缩短PMSG刺激时间对卵母细胞发育的影响。结果 将
PMSG刺激时间从46h缩短至24h,卵母细胞获得最高核成熟率（97.6% vs 91.9%）的培养时间由14h延长至16h;缩短PMSG刺激时间,核成熟率不受影响,但能显著降低激活率（91.2% vs 37.1%）和囊胚率（20.9% vs 0.0%）。 两种方法体内成熟卵母细胞激活率均高于90%,但囊胚率差异
显著(P<0.05)。卵母细胞体外培养至24~26h时,激活率（89.5%）和囊胚率（21.9%）均达到最高点。结论 建立了一种小鼠卵母细胞体外成熟培养系统,即PMSG超排处理46h、卵母细胞培养24h,CZB（10mmol/L SrCl2）激活2.5h后采用CZB（0.5%BSA）进行胚胎培养。     

Organization of tracheal epithelium in the cartilaginous portion of adult rabbit and its persistence in organ culture

Background: The rabbit trachea provides a model system to test the physiological responses of the airways to various agents. Since three-dimensional organization may affect responses of an organ, an organ culture model was established in serum free medium. Methods: Tracheas were fixed in situ, at steps in the preparation of organ cultures, and after one day to three weeks in organ culture. Samples were examined by light microscopy and scanning and transmission electron microscopy for surface morphology, distribution of cell types, and characteristics of the epithelial cell layer. Results: The normal tracheal mucosa was discovered to consist of extensive circumferential folds in the cartilaginous portion, which were enhanced upon isolation of the trachea from the animal. The folds consisted principally of differences in epithelial cell height rather than folding of the lamina propria. Enhancement of folding upon removal of the trachea coincided with increased secretion by Clara and possibly mucous cells. In organ culture, epithelial cell height initially increased, producing tall folds and cell types were retained in normal proportions. After prolonged culture, cilia were lost but glandular secretion continued. Conclusions: Changes in the arrangement of basal cells and secretory activity in isolated trachea and in culture may give insight into the functional significance of the epithelial folds. © 1994 Wiley-Liss, Inc.     

一种分离新生小鼠肝细胞的简单方法

目的建立新生小鼠肝细胞体外分离培养方法。方法采用胶原酶消化组织块法分离新生小鼠肝细胞，倒置显微镜动态观察细胞形态特征，并进行鉴定：PAS（periodieacid-Schiff，PAS）染色观察细胞肝糖元的含量；免疫组化SP法检测细胞甲胎蛋白AFP（alpha fetal protein，AFP）的表达；生化法检测肝细胞培养上清白蛋白的含量。结果肝组织块经用胶原酶直接消化，获得的肝细胞成活率达80％以上。肝细胞培养一周后长满瓶底，呈集落生长，融合成片。培养的肝细胞PAS糖原染色阳性；AFP表达阳性；培养上清白蛋白含量至培养5～7d时迭峰值，随后又逐渐下降。结论胶原酶消化组织块法分离新生小鼠肝细胞，方法简单、高效，培养的原代肝细胞符合新生肝细胞的生物学特性。     

小鼠胚胎体外培养方法的改良

目的改进胚胎培养系统以进一步提高胚胎质量与发育潜能。方法采用内径0.21mm、外径0.28mm的塑料毛细管培养胚胎,将吸有胚胎的毛细管浸在盛有蒸馏水的烧杯内,再将烧杯放置在培养箱里的磁力搅拌器上,允许胚胎在微小的空间内发育并伴随着水的旋转而摆动,更好地模拟了胚胎在输卵管中的运动环境。结果分别对85枚、82枚2-细胞胚胎进行毛细管及微滴培养,其中毛细管培养胚胎的囊胚率以及胚胎细胞数(85.4%,57.0)显著高于微滴培养(36.5%,20.6),囊胚率差异显著(P0.05)。且接种在Matrigel基底膜上形成的内细胞团集落显著增大。结论小鼠胚胎体外培养方法的改良——毛细管培养,促进小鼠植入前胚胎的体外发育。