Nile Blue Sulfate Research Articles

Hindbrain and Cranial Nerve Dysmorphogenesis Result from Acute Maternal Ethanol Administration

Acute exposure of mouse embryos to ethanol during stages of hindbrain segmentation results in excessive cell death in specific cell populations. This study details the ethanol-induced cell loss and defines the subsequent effects of this early insult on rhombomere and cranial nerve development. Ethanol at a teratogenic dosage (2.9 g/kg) or a comparable volume of vehicle was administered in each of two intraperitoneal injections to pregnant C57BL/6J mice on gestational day (GD) 8, 8 h, and GD 8, 12 h (defined hereafter as GD 8.5). Ethanol-exposed GD 9 embryos, visualized in three dimensions using laser scanning confocal microscopy of LysoTracker Red fluorescence or Nile blue sulphate vital staining, displayed excessive apoptosis in the rostral hindbrain, specifically within rhombomeres 1–3, as well as in cranial neural crest cells and ectodermal placodes. Comparably treated embryos examined on GD 10.5–11 illustrated a disproportionate reduction in the length of the rostral hindbrain. Examination of plastic histological sections of GD 9 embryos and via scanning electron microscopy on GD 10 revealed deficiencies in the hindbrain, with a phenotype including abnormal rhombomere segmentation and an extremely small fourth ventricular roofplate. Whole-mount antineurofilament immunohistochemistry on GD 10.5 and GD 11 illustrated a variety of cranial nerve abnormalities ranging from fused or absent ganglia to ectopic or disorganized fibers. In addition, a delay in the development of the glossopharyngeal (IX) nerve/ganglia complex was observed. These hindbrain and cranial nerve abnormalities are discussed in the context of the genesis of human alcohol-related birth defects and neurodevelopmental disorder.

Selective Vulnerability of Embryonic Cell Populations to Ethanol‐Induced Apoptosis: Implications for Alcohol‐Related Birth Defects and Neurodevelopmental Disorder

Ethanol-induced cell death has been characterized in very few stages of embryogenesis. This investigation comprehensively maps patterns of both programmed and ethanol-induced cell death in the central nervous system and craniofacial region at 0.5-day intervals from gestational day (GD) 6.5 to 11 in mice. A teratogenic dosage of ethanol (2.9 g/kg) or vehicle was administered via two intraperitoneal injections to pregnant C57BL/6J mice at various stages of gestation. Cell death patterns were characterized using Nile blue sulfate vital staining and histological analysis of plastic sections. Confocal laser scanning microscopy of LysoTracker Red-stained specimens allowed for three-dimensional visualization of areas of apoptosis and precise sectional imaging of mouse embryos. Apoptosis was also documented using a TUNEL technique on histological sections. Normal programmed cell death in control embryos was noted in the prechordal plate region at GD 8, the neuroepithelium of the fourth ventricle and anterior neuropore at GD 9, and within the ganglia of cranial nerves V, VII-VIII, IX, and X at GD 10. Acute maternal ethanol administration 12 hr before examination resulted in a dramatic increase in apoptosis within sites of programmed cell death in the embryo. Moreover, ethanol-exposed specimens exhibited stage-dependent excessive cell death in other distinct cell populations, particularly within the developing central nervous system. Ethanol-induced apoptosis was notable as follows: GD 7.5-neuroectoderm; GD 8-neural plate and primitive streak; GD 9-alar plate and presumptive neural crest of the rostral hindbrain, especially at the mesencephalon/rhombencephalon junction; GD 9.5-10-branchial arches and rhombomeres; and GD 11-diencephalon, basal ganglia, pons, and developing cerebellum. The results of this study revealed developmental stage-specific cell populations of the developing brain and craniofacial region that are vulnerable to ethanol-induced apoptosis and provide new insight relative to the genesis of alcohol-related birth defects.

Apoptosis and Morphology in Mouse Embryos by Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy combined with a vital stain was used to study apoptosis in organogenesis-stage mouse embryos. Apoptosis has previously been visualized in whole embryos using the vital dyes acridine orange, Nile blue sulfate, and neutral red. In the present study, mouse embryos were harvested on Gestation Day 9 and stained with the vital lysosomal dye LysoTracker Red. Following incubation in the stain, embryos were fixed overnight in 4% paraformaldehyde, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. To achieve optical sectioning through embryos, it was necessary to use low-power objectives. With this procedure, the entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. Our results demonstrate specific regions undergoing programmed cell death in normal development and increased LysoTracker staining in embryos exposed to hydroxyurea. This procedure allows for the optical imaging of whole Day 9 (∼22 somites) embryos that were greater than 700 μm thick in the z axis and can be applied to studies involving neural tube formation or other aspects of organogenesis.

Three-dimensional spectra of the long-range assembly of Nile Blue sulfate on the molecular surface of DNA and determination of DNA by light-scattering

The features of the three-dimensional (3-D) emission spectrum of the long range assembly of Nile Blue sulfate (NBS) on the molecular surfaces of DNAs were discussed. It was found that the emission signals involve resonance light-scattering (RLS, λ RLS = λ ex), second order light-scattering (SLS, λ SLS = 2 λ ex), anti-second order light-scattering (ASLS, λ ASLS = 0.5 λ ex), Raman light-scattering (Raman, λ RLS R = −49.0 + 1.28 λ ex, λ SLS R = −52.0 + 0.64 λ ex, and λ ASLS R = −171.7 + 2.86 λ ex), and fluorescence ( λ ex = 545.0 nm, λ em = 610.0 nm). The wavelengths of all light-scattering signals keep linear relationships with at of the incident light beam ( λ ex), and the intensities of the light-scattering signals in the 3-D spectrum are in the order: I RLS > I SLS > I ASLS > I RLS R > I SLS R > I ASLS R. At pH 7.20–7.80 and ionic strength 0.012, these signals, including RLS, SLS, and ASLA, were found to be strongly enhanced because of the long range assembly of NBS on the molecular surface of both calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA). Fluorescence quenching of NBS by DNAs occurs, but no significantly enhanced Raman light-scattering signals of NBS can be detected in the long range assembly. By independently using the enhanced intensity of RLS at 293.8 nm ( λ ex = 293.8 nm), ASLS intensity at 293.8 nm ( λ ex = 587.6 nm), or SLS intensity at 587.6 nm ( λ ex = 293.8 nm), ctDNA and fsDNA at nanogram levels can be determined with identical results.

Spectrophotometric study on the supramolecular interactions of nile blue sulphate with nucleic acids

The supramolecular interaction of nile blue sulphate (NBS) with nucleic acids was studied by investigating the characteristics of the interaction absorption spectra on the basis of the drug binding process in organic system in which small amount of drug interacting with large amount of biological macromolecules involves, and an accordingly binding model for organic dyes with large amount of macromolecules was established. At pH 7.40 and ionic strength 0.004, the H-aggregation of NBS occurs with increasing NBS concentration. The NBS aggregates can be bound to both calf thymus DNA and fish sperm DNA by the ratio of each nucleotide residue with a molecule of NBS if the concentration of DNAs is more than 15-fold excessive. The corresponding binding constant for the interaction of NBS with DNAs is about 10 3 order, with which thermodynamic parameters for the interactions, such as the change of free energy, enthalpy and entropy at 25°C, were calculated. It was found that the binding of NBS with thermally denatured DNA is similar to that with native yeast RNA, which indicates H-aggregation of NBS can be encouraged by single stranded nucleic acids.

Interactions of Nile Blue Sulphate with Nucleic Acids as Studied by Resonance Light-Scattering Measurements and Determination of Nucleic Acids at Nanogram Levels

ABSTRACT The interactions of nile blue sulphate (NBS) with nucleic acids, including calf thymus DNA, fish sperm DNA and yeast RNA, were characterized with resonance light-scattering (RLS) measurements by using a common spectrofluorometer. Accordingly a method for the determination of nucleic acids at nanogram levels was established. At pH's of 7.20∼7.60 and ionic strengths lower than 0.012, the interactions of NBS with nucleic acids result in three characteristic RLS peaks at 293.4 nm, 349.4 nm and 560.4 nm. Mechanism study shows that these peaks are ascribed to the long range assembly of NBS on the molecular surface of nucleic acids, which depends on pH, ionic strength and the stranded structure of nucleic acids. A Scatchard plot was constructed by using the RLS data, yielding the assembly number and assembly constant being 6.4 and 7.13x106 mol−1 1 for NBS assembly on the molecular surface of calf thymus DNA. The same parameters are 6.6 and 4.58x106 mol−1 1 for the assembly on that of fish sperm DNA, 3.9 and 1.67x106 mol−1 1 on that of yeast RNA, respectively. Linear relationships were found between the enhanced RLS intensity at 293.4 nm and nucleic acid concentration. If 1.2x10−5 mol I−1 NBS was employed, 0∼0.80 μg ml−1 calf thymus DNA and fish sperm DNA, 0.20∼0.60 μg ml−1 yeast RNA can be determined with the determination limits being 3.2 ng ml−1 for calf thymus DNA, 11.5 ng ml−1 for fish sperm DNA and 38.3 ng ml−1 for yeast RNA, respectively. Four synthetic samples were determined with satisfaction.

Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos.

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mouse embryos were harvested on gestation day 8 or 9 and stained with the vital lysosomal dye, LysoTracker Red. Following incubation in the stain, embryos were fixed in 2% paraformaldehyde overnight, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. The entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. To test this approach, the chemotherapeutic drug hydroxyurea was added to day 8 embryos in vitro to induce apoptosis. Our results demonstrated specific regions undergoing programmed cell death in normal development and increased apoptosis in embryos exposed to hydroxyurea. The observed patterns of LysoTracker Red staining correlate well with previous studies of cell death using other lysosomotropic dyes such as Nile blue sulfate, acridine orange, or neutral red. LysoTracker Red has the advantages of being aldehyde-fixable and highly fluorescent (bleaching was not observed even after multiple scans). This procedure allows for the optical imaging of whole day 9 (approximately 22 somites) embryos that were greater than 500 microns thick in the Z-axis.

Lipase production by Aspergillus and Penicillium species.

Forty each of aspergilli and penicillia were screened for extracellular lipase production on agar plates and in liquid medium containing olive oil as substrate. Twenty-nine aspergilli and twenty-six penicillia produced lipase. Out of these, 19 aspergilli and 22 penicillia showed activity both on Nile blue sulfate and glycerol tributyrate agar plates while only 10 aspergilli and 4 penicillia showed a positive response to glycerol tributyrate agar alone. The screening revealed 11 Aspergillus spp. and 15 Penicillium spp. as new lipase producers. Pig fat as an economic substrate for lipase production was also investigated.

Cell Death in the Avian Sclerotome

In this study the occurrence of apoptotic cells in chick embryo trunk somites, between 2.5 and 4 days of development, has been examined using anin situnick-end-labeling method (TUNEL) to identify nuclei in which DNA is undergoing fragmentation. At 2.5 days of development, apoptotic cells were found in the sclerotome with a distribution that depended on the rostrocaudal level in the trunk. At the most rostral levels (somites 1–18), dying cells were present primarily in the rostral half of the ventral sclerotome; at midlevels (somites 19–26), they were present throughout the ventral sclerotome; and at caudal levels (somites 27–32), no dying cells were present. By 4 days of development, the number of dying cells in the sclerotome was sharply reduced, and those present were primarily distributed to the caudal side of the intrasclerotomal fissure. Double labeling of cells for both TUNEL and the HNK-1 epitope, at 2.5 days, indicated that the majority of the dying cells were not neural crest cells. Further, dying cells in the rostral somite half were present largely in regions of the sclerotome that labeled poorly for HNK-1. It was confirmed that apoptotic neural crest cells retain the HNK-1 epitope and therefore would have been observed if present. Neural crest cells only appeared to be apoptotic in relatively small numbers and only at the ventral border of the sclerotome. Examination of DiI-labeled neural crest cells confirmed that the dying cells in the body of the somite were not primarily neural crest cells. Two hypotheses regarding the TUNEL-positive cells in the sclerotome were experimentally tested. First, that they originate from the somitocoel compartment of the somite, because their distribution patterns at 4 days were similar to those of somitocoel cells. To test this, somitocoel cells were labeled with carboxyfluorescein and grafted into host embryosin ovo. Results showed that these cells did not become apoptotic and that the dying cells were therefore not derived from the somitocoel. Second, the hypothesis was tested that the distribution patterns of the dying cells in the sclerotome are determined by factors outside the somite itself. Somites and segmental plates were transplanted into hostsin ovowith reversed orientation, after which the patterns of dying cells were examined using nile blue sulfate staining. The results indicated that the patterns were unchanged after a further 2 days incubation, suggesting that the patterns of cell death in the sclerotome are not determined solely from within the somite. The distribution of the cell death-associated gene products, bcl-2, bax, and interleukin-1β converting enzyme, indicates that although these proteins are segmentally distributed in the dermomyotome and in the rostrodorsal quadrant of the sclerotome, their patterns are not directly correlated with the distribution of dying cells.

Pathogenesis of ectrodactyly in the Dactylaplasia mouse: aberrant cell death of the apical ectodermal ridge.

Dactylaplasia, or Dac, was recently mapped to the distal portion of mouse chromosome 19 and shown to be inherited as an autosomal semi-dominant trait characterized by missing central digital rays. The most common locus for human split hand split foot malformation, also typically characterized by missing central digital rays, is 10q25, a region of synteny to the Dac locus. The Dac mouse appears to be an ideal genotypic and phenotypic model for this human malformation syndrome. Several genes lie in this region of synteny, however, only Fibroblast Growth Factor 8, or Fgf-8, has been implicated to have a role in limb development. We demonstrate that the developmental mechanism underlying loss of central rays in Dac limbs is dramatic cell death of the apical ectodermal ridge, or AER. This cell death pattern is apparent in E10.5-11.5 Dac limb buds stained with the supravital dye Nile Blue Sulfate. We demonstrate that Fgf8 expression in wild type limbs colocalizes spatially and temporally with AER cell death in Dac limbs. Furthermore, in our mapping panel, there is an absence of recombinants between Fgf-8 and the Dac locus in 133 backcross progeny with a median linkage estimate of approximately 0.5 cM. Thus, our results demonstrate that cell death of the AER in Dac limbs silences the role of the AER as key regulator of limb outgrowth, and that Fgf-8 is a strong candidate for the cause of the Dac phenotype.

Histological and histochemical studies on the paraoesophageal glands in cercariae and metacercariae of Echinostoma revolutum and E. trivolvis

AbstractHistological and histochemical studies were made on the excysted metacercariae and cercariae of Echinostoma revolutum and E. trivolvis. The paraoesophageal glands of the cercariae of both echinostome species stained positively with the intravital dyes, neutral red and nile blue sulphate. The paraoesophageal glands of excysted metacercariae of E. revolutum did not display positive staining with the intravital dyes. Some excysted metacercariae of E. trivolvis displayed positive staining of the paraoesophageal glands, while others displayed either a negative reaction or reduced staining. The lack of staining with intravital dyes, of paraoesophageal glands of excysted metacercariae of E. revolutum, suggests that the glands play a role in either the final stages of encystment, or in excystation in E. revolutum.

Effects of 2-methoxyethanol on mouse neurulation.

The potent developmental toxicant, 2-methoxyethanol (2-ME), elicits exencephaly in near-term mouse fetuses following a single maternal treatment early on gestation day (gd) 8. Deleterious morphological consequences to the neurulating embryo shortly after exposure have not been reported. The present study was designed to fill this gap and to investigate the impact of 2-ME treatment on cell death patterns in the embryonic neural folds. Dams were injected subcutaneously with saline, 250 or 325 mg 2-ME/kg 2 hr prior to the beginning of gd 8. The effect of 2-ME on gross and microscopic neural development was examined in conceptuses on gd 9, 6 hr (9:6), 10:6, and 18:0. Compared to saline, 2-ME treatment increased the percentage of embryos with open neural tubes (ONTs) at all gestation days. Although few statistically significant differences (P < 0.05) existed among the ONT rates on the 3 observation days, an interesting biological response occurred. Both high and low 2-ME doses appeared to elicit the greatest incidence of neural tube patency on gd 9:6 (affecting approximately 27% of embryos). During the subsequent 24 hr, recovery occurred and many neural folds apparently closed. Consequently, the ONT incidences on gd 10:6 (approximately 11%) were quite similar to the gd 18 exencephaly rates elicited by both chemical treatments (approximately 15%). A dose response was not seen due to a substantial increase in resorption rates following the 325 mg/kg dose. Compared to the other treatment groups, the low 2-ME dose significantly inhibited embryonic growth as indicated by reduced crown-rump and head lengths and increased incidence of developmentally delayed brain maturation. To evaluate chemically induced changes in cell death, neurulating embryos were collected on gd 8:6 and either immersed in the vital dye, Nile blue sulfate (NBS), or processed for histopathology. In 2-ME-exposed embryos, excessive NBS uptake occurred in neural fold neuroepithelium at sites of nonclosure. Using histopathology, the extent of cell death in the cephalic neural folds was dependent on the 2-ME dose, and the neuroepithelium was more severely affected than the mesenchyme. These observations suggest 1) a trend toward repair and catch-up growth later in gestation which may ameliorate the overt early effects of 2-ME, and 2) an association between enhanced cell death and regions of the neural tube particularly vulnerable to nonclosure.

Sequential changes of programmed cell death in developing fetal mouse limbs and its possible roles in limb morphogenesis.

Apoptotic cell death in the developing limb of mouse fetuses was examined sequentially on days 11-15 of gestation by means of Nile blue (NB) sulfate staining with special reference to its relation to limb morphogenesis. With some exceptions, programmed cell death (PCD) in the hand and foot was observed in the mesenchyme but not in the surface ectoderm. We found that during digital formation PCD begins at the proximal portion of the interdigital mesenchyme and subsequently expands distally. Therefore, the initial PCD that occurs in the interdigital zones may determine the proximal ends of digital separation and also contribute to the demarcation between the palm (sole) and digits (toes). During digital separation, the areas of PCD in the interdigital zones were found to become larger and expand distally on day 13, which may be necessary for the separation of digits and for determining the interdigital area to disappear. PCD in presumptive phalangeal joints was also found to proceed from proximal to more distal joints. The PCD in presumptive joints may be required for the separation of phalanges and metacarpal (metatarsal) bones and for the formation of joint cavities. In addition, intense PCD was observed in the radial (tibial) and ulnar (fibular) margins of the hand and foot plates for 4-5 days. Such PCD at marginal areas seems to prevent the formation of supernumerary digits (preaxial and postaxial polydactyly) and other digital malformations. Therefore, the timing when PCD commences and ends, the sites where PCD occurs, and the intensity, duration, and proximo-distal progress of PCD appear to be genetically determined, and the elimination of unnecessary cells by PCD may be essential for normal limb morphogenesis. The present findings also suggest that the normal progress of PCD in the hand and foot plates of rodent fetuses may prevent the formation of some limb malformations such as webbing fusion of digits, polydactyly, or cleft hand/foot.

Effect of tryptophan metabolites on fluorescent granules in the Malpighian tubules of eye color mutants of Drosophila melanogaster.

Fluorescent granules that are stained with Nile blue sulfate are present in larval Malpighian tubules in the wild type strain of Drosophila melanogaster, Oregon-R. These granules emit a weak blue fluorescence and most of them are about 2 microns or more in diameter. The ommochrome precursor 3-hydroxy-kynurenine (3-HK) is actively transferred into the tubules of Oregon-R. Changes in the fluorescent granules in the Malpighian tubules on administration of ommochrome precursors were investigated in eye color mutants of Drosophila. The fluorescent granules in the tubules of the nonautonomous mutants v;bw and cn bw emit a strong blue fluorescence and most of them are about 1 micron or less in diameter. When v;bw and cn bw larvae were cultured on medium supplemented with kynurenine or 3-HK, respectively, the fluorescence intensity of their granules decreased, and their size increased. These additions resulted in almost equal accumulation of 3-HK to that in Oregon-R. On the other hand, no 3-HK accumulated in the tubules of larvae of the autonomous mutants bw;st, ltd bw and w, which lack the fluorescent granules. These findings indicate that the fluorescent granules are an important intracellular site for uptake or storage of ommochrome precursors in larval Malpighian tubules of Drosophila.

Apoptosis induced in cultured rat embryos by intra-amniotically microinjected sodium nitroprusside.

Previously, we reported that massive cell death was induced in the mesencephalic area of cultured rat embryos after embryos of gestational day 10.5 were intra-amniotically microinjected with sodium nitroprusside (SNP, 800 microM) and cultured for 24 hr at 37 degrees C. The massive cell death apparently was the result of NO-mediated embryotoxicity. Damage was concentration dependent and tissue specific. In follow-up studies, we now report evidence that NO generated from SNP induces apoptosis in organogenesis stage cultured rat embryos. Nile blue sulfate (NBS) staining suggested that microinjections of 400 microM SNP induced apoptosis in the mesencephalic area. Since we observed no massive cell death ("white caps") at this concentration, it appeared that early stages of apoptosis preceded "white cap" formation. At 800 microM SNP, total disintegration of cell bodies was evident and may have resulted from later stages of aoptosis or necrosis, or both. The "white caps" per se, an accumulation of disintegrated cell bodies, did not stain with NBS, probably due to total loss of cell integrity and resultant coagulation. The majority of the coagulated dead cells in the "white caps" were heavily stained with 3,3'-diaminobenzidine via in situ 3' end-labeling with terminal transferase. However, it is now known that NO can damage DNA directly and that in situ 3' end-labeling by terminal transferase detects not only apoptosis but also random DNA breakage. Increased 3' end-labeling and a "DNA ladder" were detectable within 5-10 hr after exposure of day 10.5 embryos to 400 or 800 microM of microinjected SNP. Some smear background was also observed in the "ladder." Rostral aspects of embryos exhibited more prominent indices of apoptosis than caudal regions. The results suggested that microinjections of SNP into the amniotic fluid of day 10.5 cultured rat embryos induces NO-mediated cell death in the mesencephalic and rhombencephalic regions by the process of apoptosis or of both apoptosis and necrosis, depending on the timing, concentration, and stage of gestation.

Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis-stage rat embryos: effect of varying duration of deficiency.

Zinc deficiency is teratogenic in all species in which it has been examined. Zinc is an essential component of enzymes involved in DNA synthesis and cell proliferation, and may play an as yet undetermined role in apoptosis. To further our understanding of the role of zinc in normal development, we examined cell death and cell cycle parameters in embryos of pregnant rats fed a zinc-deficient diet for 2 to 10 days (0.5 microgram zinc/g diet; zinc-adequate diet was 25 micrograms zinc/g). To elucidate sensitive periods of development and susceptible cell populations, dams were fed the zinc-deficient diet from gestation day 1, 3, 7, or 9 and killed on day 11. Embryos were examined for morphology and developmental stage. From each litter, 2-3 embryos were stained with Nile blue sulfate (NBS) to visualize cell death, 3 embryos were frozen for flow cytometric cell cycle analysis and cell counts, and selected embryos were preserved for histological examination. Dams fed the zinc-deficient diet for more than 3 days reduced their food intake through gestation day 8 but increased food intake on day 9. Maternal plasma zinc dropped to 10-25% of control levels in the zinc-deficient groups. Zinc deficiency from gestation day 1 or 3 resulted in two categories of affected litters on day 11. One category had embryos which were morphologically normal but displayed extensive NBS staining in the visceral arches, neural tube, and somites. The second category had developmentally retarded or maldeveloped embryos which showed little NBS staining. Zinc deficiency from gestation day 7 produced cell death in the posterior dorsal midline in the area of premigratory neural crest cells, which was confirmed by histological examination. Zinc deficiency from gestation day 9 did not affect morphology or NBS staining. Percentages of cells in the G0/G1, S, and G2M phases of the cell cycle on gestation day 11, determined by flow cytometry, were similar to controls in all groups. This study shows that as few as 4 days of maternal zinc deficiency can produce excess embryonal cell death, and that neural crest cells may be particularly sensitive.

Programmed cell death and N-acetoxy-2-acetylaminofluorene-induced apoptosis in the rat embryo.

N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) is an alkylating agent that forms DNA adducts at C-8 in guanine and causes single strand breaks. It has previously been shown to be embryotoxic, but the mechanisms by which it causes abnormal development have not been investigated. Previous studies have indicated that other DNA alkylating agents cause cell death during embryonic development although the types of cell death were not characterized. Using a whole embryo culture system, gestation day 10 rat embryos were exposed to several concentrations (5, 50, and 200 micrograms/ml) of N-Ac-AAF. At several time points after exposure was begun (5, 10, and 24 hours), the embryos were removed from culture and examined to identify location, type and quantity of cell death, relative to programmed cell death observed in control embryos. Vital staining with Nile blue sulphate revealed that the location of N-Ac-AAF-induced cell death included the forebrain region, tail, and areas of programmed cell death. Examination of tissue sections from both control and treated embryos indicated that the location of apoptotic cell death revealed by in situ DNA nick end-labelling was generally consistent with the cell death pattern observed by vital staining of whole embryos. Agarose gel analyses indicated that all concentrations of N-Ac-AAF caused DNA fragmentation, and quantification demonstrated a dose response. Examination of treated embryos (50 and 200 micrograms/ml) by transmission electron microscopy revealed that, by 5 hours after exposure, cells with classic, ultrastructural features of apoptosis were present. In conclusion, multiple methods have all indicated that, regardless of exposure level, apoptosis was the predominant form of cell death. Because apoptosis also occurs in developmental cell death, it is possible that apoptosis induced by N-Ac-AAF is due to an alteration in cell fate via premature or ectopic induction of the cell death program.

Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation

Programmed cell death is an essential event during mammalian morphogenesis which eliminates unnecessary cells to accomplish histogenesis and organogenesis. Cell death in interdigital spaces of the developing limb is a classical example of morphogenetic cell death. We investigated whether classical programmed cell death in the interdigital tissue of the developing limb in mice is apoptosis with fragmentation of nuclear DNA and also examined sequentially the occurrence of programmed cell death and cell proliferation in the developing limb of mouse fetuses to analyze their interrelation. We examined the occurrence of apoptotic cell death in the developing limbs of mouse fetuses by using Nile blue sulphate staining, agarose gel electrophoresis for detecting DNA laddering, and a cytochemical labeling of DNA fragmentation. We also labeled proliferating cells using BrdU/anti-BrdU immunohistochemistry and examined the interrelation between apoptotic programmed cell death and cell proliferation. DNA ladders, a biochemical evidence of apoptosis, were detected in DNA extracts from the interdigital tissue of day 13 mouse fetuses by agarose gel electrophoresis. Programmed cell death and DNA fragmentation were detected by Nile blue staining and cytochemical labeling of DNA fragmentation, respectively, in the interdigital mesoderm and in the regions of presumptive joints of the digit. BrdU/anti-BrdU immunohistochemistry for identifying proliferating S-phase cells revealed that interdigital mesenchymal cells cease DNA synthesis before programmed cell death and DNA fragmentation begin. We confirmed that both cytological apoptotic alterations and fragmentation of nuclear DNA occur in the interdigital tissue and presumptive joint areas of fetal mouse limbs, and they appear to play a significant role in the separation of digits as well as the formation of joint cavities.

Embryonic cell death patterns associated with nitrofen-induced congenital diaphragmatic hernia

The offspring of pregnant Sprague-Dawley rats exposed to nitrofen on gestational day 9.5 develop left-sided congenital diaphragmatic hernia (CDH). Twenty-four hours after treatment, on day 10.5, supravital staining with Nile blue sulfate and histological examination showed bilateral excessive cell death in cervical somites 2 through 4. After 48 hours, on day 11.5, cell death was absent in the cervical somites but was apparent in the mesoderm adjacent to the somites in the septum transversum and in the developing sympathetic ganglia adjacent to the dorsal aortae. Cell death was not apparent in the foregut or lung primordia on either day 10.5 or 11.5. The incidence of nitrofen-exposed embryos with such patterns of cell death closely paralleled that of left-sided CDH in similarly treated day 21.5 fetuses. Control animals treated with olive oil had normal programmed cell death patterns in the regions of interest and had no evidence of CDH on day 21.5. It is possible that these patterns of excessive cell death early in gestation may play a role in the genesis of diaphragmatic hernia. Mesoderm derived from cervical somites 3 through 5 contributes to the diaphragmatic anlage and forms the major portion of the muscle of the diaphragm. Because nitrofen damages mesodermal cell populations in cervical somites 2 through 4 and in the mesenchyme adjacent to the septum transversum 24 to 48 hours after administration, the authors propose that damage to these populations may reduce progenitor cells needed to populate the diaphragmatic anlage, thereby hindering pleuroperitoneal canal closure. Similar embryonic mesodermal perturbations caused by unknown genetic and/or environmental phenomena may underlie the embryogenesis of human CDH.

Maternal zinc deficiency, but not copper deficiency or diabetes, results in increased embryonic cell death in the rat: implications for mechanisms underlying abnormal development.

The mechanisms underlying the teratogenicity of maternal copper deficiency, zinc deficiency, and diabetes are largely unknown. Here we investigated whether these insults are associated with altered patterns of cell death in gestation day (GD) 11.0 rat embryos. Four weeks prior to mating, rats in the copper-deficient group (CuD) were fed a copper-deficient diet supplemented with the chelator, triethylenetetramine, to facilitate the depletion of tissue copper stores. Rats in this group were switched to a triethylenetetramine-free copper-deficient diet 1 week prior to mating. Dams in the diabetic and control groups were fed a control (8 micrograms copper, 25 micrograms zinc/g) diet throughout the study. On GD 3.0, one subset of the control dams was assigned to the zinc-deficient group (ZnD) and fed a zinc-deficient diet. A second subset of control dams was assigned to a restricted fed group and fed the control diet in quantities consumed by the zinc-deficient dams. Litters were taken by cesarean section on GD 11.0. Embryos were examined for gross morphology and assessed for patterns of cell death using Nile blue sulfate. Embryos from the CuD dams were characterized by edematous hindbrain. Embryos from the diabetic group were characterized by delayed development. Altered patterns of cell death were only detected in embryos from the ZnD dams. Within the ZnD group, embryos were either characterized by small size, edematous head region, and control patterns of cell death, or normal size, normal morphology, and increased cell death. These different patterns of morphology and cell death in the embryos of ZnD dams were associated with different patterns of maternal food intake.(ABSTRACT TRUNCATED AT 250 WORDS)