Method Of Whole-embryo Culture Research Articles

Morphological phenotyping after mouse whole embryo culture.

Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between embryonic days 8.5 and 9.5. During this period, neural tube closure is initiated and progresses to completion in the cranial region. Spinal closure is still underway at the end of the culture period. The focus of this article is particularly on phenotyping that can be performed at the bench, using a stereomicroscope. This involves assessment of embryonic health, through observation and scoring of yolk sac blood circulation, measurement of developmental stage by somite counting, and determination of crown-rump length as a measure of growth. Axial rotation ("turning") can also be assessed using a simple scoring system. Neural tube closure assessment includes: 1) determining whether closure has been initiated at the Closure 1 site; 2) evaluating the complex steps of cranial neurulation including initiation at Closure sites 2 and 3, and completion of closure at the anterior and hindbrain neuropores; 3) assessment of spinal closure by measurement of posterior neuropore length. Interpretation of defects in neural tube closure requires an appreciation of, first, the stages that particular events are expected to be completed and, second, the correspondence between embryonic landmarks, for example, somite position, and the resulting adult axial levels. Detailed embryonic phenotyping, as described in this article, when combined with the versatile method of whole embryo culture, can form the basis for a wide range of experimental studies in early mouse neural development.

Embryotoxicity of Transgenic Rice TT51 and Cry1Ab Bt Insecticidal Toxin in Rat Post Implantation Whole Embryo Culture

TT51, with a synthetic Cry1Ab/CryAc gene from Bacillus thuringiensis Berliner, was the first genetically modified (GM) plant officially certificated in China. This study was undertaken to investigate potential embryotoxicity of TT51 on rats. Whole embryo culture (WEC) is an effective method for safety evaluation when culture medium, containing substances derived from GM food, is available. Rat embryos explanted from uterus were randomly distributed into 6 groups: Transgenic Rice (TR) group with transgenic rice dietary administration, Parental Rice (PR) group with parental rice dietary administration, Blank Control (BC), Positive control A (PCA) group with Bisphenol A being directly added into the culture medium, Positive control B (PCB) group with Bisphenol A oral administration and Bt protein (BP) group with Bt protein directly added into culture medium. After culturing for 48 h, embryos were scored for growth and differentiation at the endpoint. Embryos in TR group had no significantly lower morphological scores or smaller yolk sac diameter, crown-rump length (CRL) and cranial diameter comparing with those in BC and PR group. Embryos in PCA and PCB group both performed significantly lower developmental parameters and morphological scores than the other three groups. Embryos directly exposed to Bt protein (52 mg/L) exhibited severe morphological anomalies but no significant difference in yolk sac diameter compared with PR, TR and BC groups. In this WEC model, TT51 showed no embryotoxicity to rats although Bt toxin had side effect on embryos when the concentration is equal to the daily intake of Bt protein in TT51 diet. And it also indicated that WEC method can make an option for toxicological assessment of GM food.

Alternative Splicing of the Robo3 Axon Guidance Receptor Governs the Midline Switch from Attraction to Repulsion

Alternative splicing provides a means to increase the complexity of gene function in numerous biological processes, including nervous system wiring. Navigating axons switch responses from attraction to repulsion at intermediate targets, allowing them to grow to each intermediate target and then to move on. The mechanisms underlying this switch remain poorly characterized. We previously showed that the Slit receptor Robo3 is required for spinal commissural axons to enter and cross the midline intermediate target. We report here the existence of two functionally antagonistic isoforms of Robo3 with distinct carboxy termini arising from alternative splicing. Robo3.1 is deployed on the precrossing and crossing portions of commissural axons and allows midline crossing by silencing Slit repulsion. Robo3.2 becomes expressed on the postcrossing portion and blocks midline recrossing, favoring Slit repulsion. The tight spatial regulation of opponent splice variants helps ensure high-fidelity transition of axonal responses from attraction to repulsion at the midline.

Expression of glucose-regulated proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro.

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.

Environmental and genetic determinants of neural migration and postmigratory survival.

The study of genetic/epigenetic/environmental factors underlies all therapeutic and preventive approaches in fetal, perinatal and paediatric neurology, including rehabilitation. In this paper, we selected a few targets of environmental determinants of brain development leading to underlying priorities for protection of the developing brain. Preparation of the neural germinative epithelium has to be protected against noxious pharmacological agents. New tools have been developed to improve early neural teratology, including the whole-embryo culture method. The neopallial astrocytic precursors have a dual origin. Astrocytes of the white matter and deep neocortical layers derive from transformed radial glial cells, whereas astrocytes of the upper neocortical layers derive from astrocytic precursors that migrate from the late germinative zone after the end of neuronal migration. Among numerous factors able to interfere with these gliogenetic events are the control factors of the lysosomal and autophagic functions, interfering with radial glial cell transformation into astrocytes. All lesions interrupting the migratory corridors of late astroglial migration can produce cytoarchitectonic disturbances of the neocortical supragranular layers, with long-term consequences. The developing brain is weltering in a complex mixture including newly recognized excitotoxic substances, cytokines and growth factors. These substances are sometimes environmental friends like maternal vasointestinal peptide, which prevents brain intrauterine growth retardation. They are sometimes excellent endogenous friends like neurotrophic excitatory agents in physiological conditions. They become often dangerous killers triggered by environmental signals like hypoxias/ischaemias and toxins produced by intrauterine infections, launching the excitotoxic cascade. In this paper, we reviewed mainly environmental determinants interfering with neural cytogenesis and histogenesis during the embryonic, fetal and neonatal span of early life.

Mechanisms of normal and abnormal neurulation: evidence from embryo culture studies.

The method of whole embryo culture has been used extensively in analyzing the mechanisms underlying formation of the mammalian neural tube. These studies have provided insight into the cell lineage of the various tissues that comprise the neurulation stage embryo, the role of microfilaments, extracellular matrix and cell proliferation in the morphogenetic events of neural tube closure and the action of specific genes and gene products in establishment of the nervous system. This information is of considerable importance not only as a means of elucidating the processes of normal embryogenesis but also to shed light on the pathogenesis of important human birth defects.

DYSMORPHOGENIC EFFECTS OF IVOMEC IN MOUSE EMBRYOS IN VITRO

Iverm ectin is an antiparasitic agent used in animals and humans and is often administered as a 1% solution (Ivomec, Merck) dissolved in propylene glycol and glycerol formal. Ivermectin alone produces minor adverse reactions in humans and farm animals, but mice are particularly sensitive to the drug, demonstrating neurotoxicity in adults and malformations in offspring at relatively low maternal doses. Ivom ec and its constituent solvents, propylene glycol and glycerol formal, have not been previously examined for their direct effect on embryonic development. In this study the in vitro method of whole-embryo culture was used to expose early-som ite mouse embryos to Ivomec, propylene glycol, and glycerol formal. Pure iverm ectin could not be obtained from the manufacturer and thus could not be evaluated individually. Ivomec was added to the culture medium to achieve ivermectin concentrations of 0, 5, 10, 25, and 50 ug/ ml. Controls for each group were exposed to propylene glycol and glycerol formal (no ivermectin) at the same concentrations as Ivomec-treated embryos. Em bryos were also exposed individually to propylene glycol and glycerol formal at the same concentrations used earlier: 0, 114, and 228 ug/ m l (propylene glycol) and 0, 21, 42, 104, and 208 ug/ ml (glycerol form al). Embryonic m orphology, heart rate, somite num bers, and protein levels were evaluated after 24 h in culture. Ivomec produced malform ations in early-som ite m ouse embryos in vitro in a fairly dose-dependent m anner, with malformation rates of 14% at 5 ug/ ml, 44% at 10 ug/ m l, 40% at 25 ug/ ml, and 88% at 50 ug/ m l, compared to 3% in normal controls. Corresponding controls treated with both solvents together dem onstrated a similar response, with malformation rates of 20%, 25%, 30%, and 79%, respectively, suggesting that propylene glycol and/ or glycerol formal may be largely responsible for the observed effects. When studied individually, propylene glycol was dysm orphogenic only at 228 ug/ m l, whereas glycerol form al produced dose-dependent m alform ations at 21 ug/ m l. Therefore, glycerol form al may be largely responsible for the dysm orphogenic effects of Ivom ec, but the mechanisms responsible for these effects are not yet understood.

Embryopathic effects of diazoxide and the reduction of sulfonylurea-induced dysmorphogenesis in vitro

Diazoxide is a benzothiadiazine used in oral and intravenous preparations to treat hypertension and hypoglycaemia, but its effect on embryonic development has not been well studied. Previous in vivo work has suggested placental transfer and teratogenicity of diazoxide in humans and laboratory animals, but this study represents the first in vitro investigation of the effect of diazoxide on the embryo. The in vitro method of whole-embryo culture was used to expose mouse embryos to specific levels of diazoxide (0–200 μg/ml) during organogenesis at well-defined (4–6 and 20–25 somite) stages of development. In addition, the combined effect of diazoxide [ATP-sensitive K + (K ATP) channel opener] and the sulfonylurea oral hypoglycaemic agent chlorpropamide (K ATP channel blocker), was evaluated in vitro in embryos with 4–6 somites. Diazoxide produced malformations and growth retardation in mouse embryos exposed to 100 μg/ml or more for 24 hr in vitro at both stages of organogenesis. In addition, a subteratogenic concentration of diazoxide (25 μg/ml) reduced the embryopathic effects of chlorpropamide (130 μg/ml) in embryos with 4–6 somites. A potential mechanism for these effects involves K ATP channels.

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.

Embryotoxic effects of chlorobutanol in cultured mouse embryos.

Chlorobutanol (CB) is a commonly used preservative which is added to numerous pharmaceutical preparations, and it is the active ingredient in certain oral sedatives and topical anesthetics. Chlorobutanol has demonstrated adverse effects in adult tissues, but CB has not been previously investigated for its effect on the developing whole embryo. The method of whole-embryo culture was used in this study to expose mouse embryos during two stages of organogenesis to CB at final concentrations of 0 (control), 10, 25, 50, 100, and 200 micrograms/ml. Embryos were evaluated for heart rate (HR), malformations, and somite number, and embryos and visceral yolk sacs (VYSs) were assayed for total protein content as a measure of overall growth. Neurulating (3-6 somite) embryos were malformed and growth retarded by exposure to CB concentrations > or = 25 micrograms/ml, with decreased VYS growth at > or = 50 micrograms/ml and decreased HR at > or = 100 micrograms/ml CB. Early limb-bud stage (20-25 somite) embryos were malformed at CB concentrations > or = 50 micrograms/ml and growth retarded at > or = 100 micrograms/ml, with decreased VYS growth at 200 micrograms/ml and decreased HR at > or = 100 micrograms/ml CB. Thus, CB produces dysmorphogenesis in mouse embryos in vitro, and neurulating embryos are somewhat less sensitive than early limb-bud stage embryos. The concentrations of CB that interfere with normal embryonic development are within the range of human blood levels measured following multiple doses of CB. Preparations containing CB should be used with caution during pregnancy, particularly when repeated dosing may allow accumulation of CB to potentially embryotoxic levels.

Transfer of plutonium to rat embryos in vivo and in vitro.

The 239Pu distribution in the 12.5-day-old rat conceptus was compared between in vivo and in vitro experimental systems to establish a possible mechanism of cross-placental transfer of this radionuclide. In the in vivo study, plutonium citrate solution was injected intravenously to pregnant Wistar rats. In the in vitro study, either plutonium citrate or plutonium hydroxide colloid was administered, as a solution of Eagle MEM and FCS containing 239Pu at the concentration used in the maternal serum in the in vivo experiments, to rat conceptuses maintained by the whole-embryo culture method. The concentration of 239Pu in the yolk sac (239Pu activity per gram wet weight) were much higher than in the embryo in both the in vivo and in vitro experiments, suggesting that the yolk sac may be an effective barrier against the transfer of plutonium to the embryos. The ratios of the 239Pu concentration in the yolk sac to that in the embryo were relatively constant with time after administration in the in vitro system; 18-27 for plutonium citrate and 67-84 for plutonium hydroxide. In the in vivo experiment, these ratios changed with time after injection; 15 at 5 min and 62 and 60 min after injection. This suggests that in the in vivo system, the chemical form of 239Pu changed with time after injection, probably to a macromolecular form such as the hydroxide colloid or plutonium-protein complex although 239Pu was injected to the maternal blood as citrate.

Glycosaminoglycans vary in accumulation along the neuraxis during spinal neurulation in the mouse embryo

We have utilized the method of whole embryo culture for metabolic labeling of mouse embryos with [ 3H]glucosamine during closure of neural folds at the posterior neuropore (27- to 29-somite stage). Accumulations of newly synthesized glycopeptides, lactosaminoglycans, hyaluronate, and sulfated glycosaminoglycans (GAG) were assessed by ion-exchange chromatography of glycoconjugates isolated from labeled embryos. Accumulation of hyaluronate and sulfated GAG was greatest in the posterior neuropore and decreased progressively toward the hindbrain where neurulation was already complete. Hyaluronate comprised a progressively smaller proportion of total newly synthesized glycoconjugate from the posterior neuropore toward the cranial region and glycopeptides showed the opposite trend. Sulfated GAG and lactosaminoglycans showed no consistent differences in relative abundance along the neuraxis. Autoradiographic analysis of newly synthesized glycoconjugates revealed especially heavy incorporation into developing basement membranes, beneath the neuroepithelium and around the notochord, in the posterior neuropore and recently closed neural tube regions, but not at more cranial levels of the neuraxis. Predigestion of sections with a specific hyaluronidase showed a significant quantity of this glycoconjugate to be hyaluronate. These results are consistent with a role for neuroepithelial and notochordal basement membrane hyaluronate in spinal neurulation.

Serotonin and morphogenesis. I. Sites of serotonin uptake and -binding protein immunoreactivity in the midgestation mouse embryo.

The possible involvement of the neurotransmitter serotonin (5-HT) in morphogenesis of the craniofacial region in the mouse embryo has been investigated using the method of whole-embryo culture. Day-12 embryos were incubated for 3-4 h in the presence of 5-HT or its precursors L-tryptophan (L-TRP) or 5-hydroxytryptophan (5-HTP), followed by fixation, sectioning and staining with a specific antiserum to 5-HT. Sites of 5-HT immunoreactivity were found in a variety of locations in tissues of the head and neck, which are either epithelia derived from the non-neural ectoderm or are non-neuronal midline brain structures. These sites include the surface epithelia of the head, face, nasal prominences, branchial arches, oral cavity and associated parts of the nasal epithelium, the epithelium covering the eye, parts of the otic vesicle, the epiphysis and roof of the diencephalon. With the exception of the oral cavity, sites of immunoreactivity for serotonin-binding protein were identified in the mesenchyme adjacent to these sites. This mesenchyme consists of ectodermally derived neural crest cells, which are known to receive inductive influences from the epithelia with which they interact during their migration through the craniofacial region. The presence of 5-HT uptake sites in epithelia and adjacent sites of SBP in the underlying mesenchyme raises the possibility that 5-HT might be involved in those epithelial-mesenchymal interactions known to be important for the development of structures in the craniofacial region.

Validation of the whole-embryo culture method for in vitro tesatogericity testibg

Caffeine and chloroquine were administered to pregnant SAFICR mice at doses which were non-teratogenic. Nevertheless, these dose levels enhanced significantly the number of cleft palates produced by 200 rads x-ray. The combination of chloroquine with 200 rads x-ray also resulted in a substantially higher incidence of tail abnormalities. Although the mechanisms of action for most teratogens are undefined, mutagenic damage to DNA has been implicated. We propose a “co-teratogenic” mechanism by which non-teratogenic agents enhance the effectiveness of gene damage in producing fetal abnormalties.