Forelimb Buds Research Articles

Cell cycle alterations and cell death in cyclophosphamide teratogenesis

Litters of pregnant mice treated with cyclophosphamide (CP) exhibit malformations of the limbs ranging from oligodactyly to amelia. Previous studies have indicated that cell death occurs in limb buds shortly after maternal exposure. We have investigated the relationship of cell death, cell cycle perturbation, and embryo/fetal toxicity in the mouse using vital staining and flow cytometry (FCM). CP (20, 30, and 40 mg/kg) was investigated via intraperitoneal administration to Swiss-Webster mice on day 10 of gestation. At 4, 8, or 28 hours later, embryos were removed. Cell death was identified with Nile blue sulphate (NBS). Two embryos per litter were stained with NBS, and the remaining embryos were frozen at -70 degrees C prior to FCM analysis. After thawing, the forelimb buds were removed for the isolation of nuclei. Tissues were dissociated through a wire mesh followed by cytolysis with 0.1% nonidet P-40 in PBS with 0.5 mg/ml RNase. Nuclei were stained with the fluorescent nucleic acid probe propidium iodide and analyzed (10,000 nuclei per sample) for propidium iodide fluorescence by FCM. NBS revealed a dose-related increase in cell death by 8 hours after dosing. CP-induced cell death was greatest in areas of rapid cell proliferation (DNA synthesis). FCM analysis revealed retardation of progression through the S-phase of the cell cycle by 4 hours post-exposure at all doses. This retardation occurred earlier in S-phase with increasing dose and persisted through 8 hours. At 28 hours, cell cycle histograms were normal in the low-dose embryos, but remained perturbed in the intermediate- and high-dose embryos. On day 17 of gestation, the last group of dams was killed. A high incidence of fetal malformations, including limb defects, occurred at the 20 mg/kg dose, and fetal mortality was observed at 30 and 40 mg/kg. The pattern and magnitude of cell death correlated with cell cycle perturbation and fetal toxicity at term, suggesting a relationship between cell cycle perturbation, cell death, and malformations produced by CP.

A gradient of homeodomain protein in developing forelimbs of Xenopus and mouse embryos

The expression of the homeodomain protein XIHbox 1 in developing Xenopus limbs was analyzed using specific antibodies. In the forelimb bud mesoderm XIHbox 1 shows a clear antero-posterior gradient that is strongest in the anterior and proximal region of the forelimb. Hindlimb bud mesoderm is devoid of XIHbox 1, indicating an early molecular difference between arm and leg. The innermost ectodermal cell layer is positive throughout the forelimb and hindlimb bud ectoderm, but no other areas of the skin. Similar results are obtained in developing mouse limbs, suggesting that XIHbox 1 participates in forelimb development in a variety of tetrapods. In early tadpoles analyzed at stages preceding limb bud formation, the lateral plate mesoderm is positive in the region corresponding to the earliest “field” of forelimb information, but not in the hindlimb field. These results suggest a molecular link between morphogenetic fields, gradients, and homeobox genes in vertebrate development.

Phenytoin-induced stress protein synthesis in mouse embryonic tissue.

Several proteins have been shown to be synthesized in response to various environmental stimuli, including treatment with teratogens. The role of these proteins in the teratogenic process is unknown. Pregnant A/J mice were treated with either a teratogenic or a non-teratogenic dose of the anticonvulsant drug, phenytoin (PHT). Protein synthesis in embryonic craniofacial (target) tissue or forelimb buds (non-target) was determined by incorporation of radiolabeled leucine and analysis by two-dimensional polyacrylamide gel electrophoresis. Synthesis of three proteins in target tissue and one protein in non-target tissue was stimulated by drug treatment. These results suggest that synthesis of specific stress proteins may serve as biomarkers of drug-target tissue interaction.

Innervation and behaviour of ectopic limbs in Xenopus

The hindlimb bud of Xenopus tadpoles was replaced with a forelimb bud, or vice versa, prior to axon invasion of the limb. Ectopic hindlimbs supported 94% as many brachial motoneurons as the remaining forelimb, and ectopic forelimbs supported 46% as many lumbar motoneurons as the remaining hindlimbs, on average, after the period of motoneuron death. The patterns of movement of ectopic limbs were characteristic of the innervating spinal cord segments, and not the limbs. The anatomical patterns of nerve trunks were characteristic of the ectopic limbs, and not the sources of innervation. HRP transport studies showed a resemblance between the locations of lumbar motoneurons supplying ectopic forelimb muscles and those supplying the homologous muscles of the hindlimb. It was concluded that motoneurons could survive the period of cell death following connection to muscles for which their patterns of activity were inappropriate, and the projections to ectopic limb muscles were specific although the nerve paths within the limbs were different.

Cytotoxic effects of ethylene glycol monomethyl ether in the forelimb bud of the mouse embryo.

The role of cytotoxicity in digital maldevelopment in CD-1 mouse embryos was examined following dosage with ethylene glycol monomethyl ether (EGME) on gestation day (gd) 11. Patterns of cell necrosis in the forelimb buds of embryos collected from dams given EGME orally at doses of 100, 250 or 350 mg/kg were characterized by staining with Nile blue A. Cell death was induced in the mesenchymal tissue and to some extent in the limb bud ectoderm, including the apical ectodermal ridge in a dose-related manner. The area of preaxial physiological cell necrosis was enlarged by EGME, and the shape of the limb buds was altered 24 hr after treatment. Preaxial tissue and the predigital chondrocyte condensations were reduced or missing following 250 and 350 mg EGME per 1 kg. Light and electron microscope evaluations of forelimb buds revealed the presence of phagocytic vacuoles and condensed, fragmented cytoplasm, which indicate cytotoxicity, as early as 2 hr following EGME, a maximum effect being observed 6 hr after the dose was administered. Although the severity of the cytotoxic response appeared to be dose-related, comparison with the incidence of digital malformations in near-term fetuses indicates that the loss of mesenchymal tissue is partially compensated for as formation of the limb progresses.

Neurulation in the mouse. I. The ontogenesis of neural segments and the determination of topographical regions in a central nervous system.

Ontogenesis of neural segments and positional relationships between the segments and other organs during neurulation were studied in 1,423 ICR mouse embryos by binocular dissecting, light, and scanning electron microscopy. Late in the presomite stage, two transverse sulci, preotic and otic, were seen on the prospective luminal surface of the neural folds. By somite stage 19, the former subdivided into five neuromeres, and by somite stage 21, the latter subdivided into four neuromeres. From the rostral, preotic sulcus, moreover, five other neuromeres were formed by somite stage 20, and between the otic sulcus and the first somite, two neuromeres were formed by somite stage 28. In the caudal part, from the level of the first somite, a total of 39 neuromeres were formed one after another by somite stage 39, and their positions almost correlated with each corresponding somite. Furthermore, the isthmus grew in the boundary between the fifth and sixth neuromere. The most protruding zone in the preotic sulcus formed the eighth neuromere and was located adjacent to the first branchial arch and the trigeminal ganglion. The most protruding zone in the otic sulcus also formed the 11th neuromere and was located adjacent to the second branchial arch. The 12th and 13th neuromeres were situated adjacent to the otic vesicle; the 23rd to 28th neuromeres, adjacent to the forelimb bud; and the 40th to 46th neuromeres, adjacent to the hindlimb bud.

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G 0 + G 1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.

Effect of glutathione depletion by buthionine sulfoximine on rat embryonic development in vitro

The intracellular thiol glutathione has many functions within cells including protection against xenobiotic and oxidative damage, and a role in protein and DNA synthesis and amino acid transport. Consequently, glutathione might be an important substance for normal growth and development. In this study the extent of glutathione depletion by buthionine sulfoximine, an agent which depletes glutathione by inhibiting its synthesis, and the subsequent effects of the depletion on rat embryonic growth and development were assessed. Day 10.5 rat embryos were cultured in rat serum medium in the presence of l-buthionine -S,R- sulfoximine (0.01 to 2.0 mM) and examined for viability, malformations, growth and development 45 hr later. The glutathione concentrations of the cultured embryos and their yolk sacs were also determined. Exposure to buthionine sulfoximine produced marked and significant (P ⩽ 0.05) depletion of glutathione at a buthionine sulfoximine concentration of 0.10 mM in the embryos and 0.05 mM in the yolk sacs. Exposure to 1 mM buthionine sulfoximine depleted glutathione to less than 7% of control in both of these tissues. None of the concentrations of buthionine sulfoximine tested had a significant effect on embryo viability; however, buthionine sulfoximine caused a significant (P ⩽ 0.05) incidence of malformed embryos at concentrations of 0.25, 0.5, 1.0 and 2.0 mM. The types of defects induced by buthionine sulfoximine were blebs of the maxillary or nasal processes, prosencephalon or forelimb buds, small or misshapen heads, small prosencephalons and swollen hind brains, and tail defects. Embryonic growth was the most sensitive, of the variables assessed, to the effects of buthionine sulfoximine. Significant (P ⩽ 0.05) growth retardation was observed at buthionine sulfoximine concentrations as low as 0.01 mM. At 2.0 mM buthionine sulfoximine, the yolk sac diameter, embryo crown-rump length, head length, number of somites and morphological score were reduced to 65, 72, 77, 90 and 80% of control levels respectively. We propose that the embryotoxic effects of buthionine sulfoximine are due to glutathione depletion and, consequently, that a certain basal level of endogenous glutathione is essential to allow for normal development.

New in vitro procedures for experimental studies on the development of 11-day mouse embryo forelimb buds.

A new method has been developed for culturing 11-day mouse forelimb buds in vitro. In cultures performed with conventional procedures, skeletal pieces frequently appeared distorted and reduced in size. Moreover, forelimb buds explanted from embryos younger than a stage corresponding to 50 pairs of somites developed narrow hand plates devoid of radiated autopods. By contrast, in the new procedure using media supplemented with fetal calf serum and growth factors and enhancing distal feeding with carrier implants of catgut, enlarged pads were obtained that exhibited at least 4 digital rays in buds explanted from embryos with 40-44 pairs of somites. Compared with conventional procedures, the mean value of DNA content per limb bud was twice as great with use of our improved method. The ability of limb bud cells to proliferate and differentiate when cultured either in classical or in modified conditions, and the importance of the technical procedures, are discussed in the new prospect of in vitro developmental studies.

Developmental regulation of the multiple myogenic cell lineages of the avian embryo.

The developmental regulation of myoblasts committed to fast, mixed fast/slow, and slow myogenic cell lineages was determined by analyzing myotube formation in high density and clonal cultures of myoblasts isolated from chicken and quail embryos of different ages. To identify cells of different myogenic lineages, myotubes were analyzed for content of fast and slow classes of myosin heavy chain (MHC) isoforms by immunocytochemistry and immunoblotting using specific monoclonal antibodies. Myoblasts from the hindlimb bud, forelimb bud, trunk, and pectoral regions of the early chicken embryo and hindlimb bud of the early quail embryo (days 3-6 in ovo) were committed to three distinct lineages with 60-90% of the myoblasts in the fast lineage, 10-40% in the mixed fast/slow lineage, and 0-3% in the slow lineage depending on the age and species of the myoblast donor. In contrast, 99-100% of the myoblasts in the later embryos (days 9-12 in ovo) were in the fast lineage. Serial subculturing from a single myoblast demonstrated that commitment to a particular lineage was stably inherited for over 30 cell doublings. When myoblasts from embryos of the same age were cultured, the percentage of muscle colonies of the fast, fast/slow, and slow types that formed in clonal cultures was the same as the percentage of myotubes of each of these types that formed in high density cultures, indicating that intercellular contact between myoblasts of different lineages did not affect the type of myotube formed. An analysis in vivo showed that three types of primary myotubes--fast, fast/slow, and slow--were also found in the chicken thigh at day 7 in ovo and that synthesis of both the fast and slow classes of MHC isoforms was concomitant with the formation of primary myotubes. On the basis of these results, we propose that in the avian embryo, there is an early phase of muscle fiber formation in which primary myotubes with differing MHC contents are formed from myoblasts committed to three intrinsically different primary myogenic lineages independent of innervation and a later phase in which secondary myotubes are formed from myoblasts in a single, secondary myogenic lineage with maturation and maintenance of fiber diversity dependent on innervation.

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro.

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.

Histological changes induced in developing limb buds of C57BL mouse embryos submitted in utero to the combined influence of acetazolamide and cadmium sulphate.

Microscopic defects in limb buds of C57BL mouse embryos after the combined teratogenic action of acetazolamide plus cadmium sulphate administered on day 9 of gestation were studied in serial sections. Postaxial deficiencies observed in 12-15-day embryos and affecting preferentially the right forelimbs were classified in nine morphological types according to increasing amounts of missing parts. Type X defect consists of a nearly complete amelia in which all four limbs are represented only by the girdle and proximal end of the stylopod. Type XI abnormality appears as an intermediate reduction affecting the area of digit IV. In addition to modifications of the forelimb bud shape detected from the 10-day stage onwards, observations made 24 and 48 hr after treatment confirmed that the postaxial defects result from an absolute lack of postaxial mesoderm occurring without cell necrosis as a consequence of a postaxial shortening of the apical ectodermal ridge (aer). In 10-day embryos, the latter appears shortened and hypertrophied; it is later fragmented into alternate thick and thin portions in 11-day affected limb buds. These ectodermal changes might account for the genesis of all types of defects observed. Untreated 9-day embryos with 12-25 pairs of somites display a number of asymmetries between their right and left forelimb territories: Until the 19-somite stage, the vascular supply to that area is provided exclusively by the umbilical vein, which is larger on the right side; the initial amount of somatopleural limb mesoderm is greater in the right rudiment and the genesis of its aer is slightly protracted as compared to the left one. These asymmetries might contribute to the right side predominance of the forelimb defects induced by acetazolamide and cadmium.

Pathogenesis of the mouse forelimb deformity induced by acetazolamide: an electron microscopic study.

Scanning electron microscopic observations after removal of the epidermis from developing limb buds reveal a fine mesenchymal cell process meshwork (CPM). The relationship between apical ectodermal ridge (AER) development and CPM density was investigated and related to the postaxial reduction deformities induced by acetazolamide (AA). AA was given orally to pregnant mice at 9 A.M. and 4 P.M. of day 9 and 9 A.M. of day 10 (VP = 0) in a dose of 1,000 mg/kg. Forelimb ectrodactyly, especially on the right, was the most common deformity observed. Scanning electron microscopic observations showed that the AER in AA-treated right forelimb buds did not extend postaxially as far as that in controls. The postaxial region with the hypoplastic AER became defective. Scanning and transmission electron microscopic observations revealed that in control and treated right forelimb buds, the CPM underneath the typical AER was sparser than that underneath the dorsal or ventral non-ridge epidermis. However, in treated right forelimb buds, the CPM underneath a hypoplastic AER was denser than that underneath the normal AER. These findings suggest that AA-induced deformity results from a disturbance of the AER-mesenchymal interactions.

Developmental Changes of Cell Process Meshwork and Extracellular Space in Subepidermal Mesenchyme in Mouse Forelimb Buds: (apical ectodermal ridge/mesenchymal cell process/subepidermal extracellular space/ limb bud/mouse embryo).

This study was designed to reveal morphological changes in epithelial-mesenchymal interface during limb development. An electron microscopic and morphometric analysis was done on the cell process meshwork (CPM) and subepidermal extracellular space (SEECS) in mesenchyme in mouse forelimb buds at embryonic ages from day 9.5 to 12.5 (vaginal plug=day 0). At days 9.5 and 10.0, when the apical ectodermal ridge (AER) had not yet appeared, no spatial differences were noted in the CPM density and SEECS width. However, at days 10.5, 11.0 and 11.5, when the AER was distinct, the SEECS was wide and the number of mesenchymal cell processes was small beneath the AER as compared with those at days 9.5 and 10.0; they differed significantly from those beneath the dorsal or ventral non-ridge epidermis. Between days 10.0 and 10.5, the SEECS width decreased and the number of cell processes increased in the dorsal region. At days 12.0 and 12.5, when the AER was regressing, the spatial differences in the CPM density and the SEECS width became less obvious. These findings indicate that spatial and temporal differences of the SEECS width and CPM density exist in mouse limb buds, and these differences are closely associated with the AER development.

Histogenesis in 11-day mouse embryo limb buds explanted in organ culture.

Fore- and hindlimb buds from 11-day mouse embryos with 40 to 52 somites (including the four occipital pairs) were explanted in organ culture and submitted to systematic histological analysis. Chondrogenesis occurs normally in culture in all preskeletal rudiments which were already represented by condensed blastemas before explantation. In the proximal territories, the progress of cartilage differentiation occurs according to the normal pattern and can be revealed histologically much earlier than in bulk preparations. In all explanted hindlimbs as well as in forelimbs from embryos with less than 50 somites, a primary coalescence occurs between the IId and IIId digital rays, leading to various fusions from soft tissue syndactyly to oligosyndactyly. This is the result of two combined unfavorable effects of the culture conditions: the lack of simultaneous volumetric growth of the foot- or handplate, which normally would provide the necessary space for the laying down of a pentadactylous pattern, and a loss of cells resulting from abnormal cell death affecting selective mesodermal sites in the zeugopod and in the marginal subridge area, the latter being more severely affected in hindlimb buds. Several observations suggest that the preferential sensitivity of the marginal mesoderm might be related to early changes in the apical ectoderm, which itself becomes excessively necrotic and rapidly looses its pseudostratified configuration. The forelimb buds from embryos with 50 somites and more usually develop a pentadactylous pattern with a better individuation of digital structures. In all explants, the prospective mesoderm of digit I exhibits stronger regulatory tendencies.

Accelerated maturation of limb mesenchyme by the Brachypod H mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism H ( bp H ) mutation are shown to behave similarly to ‘normal’ proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than ‘normal’ distal or whole limb bud cells. These results support the hypothesis that the bp H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.

Autoradiographic study of glycoprotein synthesis during embryonic epidermogenesis of the mouse in vitro.

Forelimb buds of mouse embryos (day 11 + 3 h of development) were cultured for 6 days. During this period, the epidermis developed from a two-layered stage (basal and periderm cell layer) to a multilayered squamous epithelium wih stratum granulosum and stratum corneum. To investigate the glycoprotein synthesis during epidermogenesis in vitro, the limb buds were labeled for 30 min or 4 h with 3H-fucose at successive stages of development. Until the development of the stratum intermedium, no different labeling of the individual layers of the epidermis and the periderm was observed. Shortly before the beginning of the stratum granulosum, a strong labeling of the outer region of the intermediate layer, where the shape of the cells had changed from cuboid to flat, was visible. When the stratum granulosum developed, even in this layer and in the upper stratum spinosum, strong glycoprotein synthesis was observed, indicated by strong labeling with 3H-fucose. In the developing stratum corneum, no labeling of this layer was detectable 30 min or 4 h after 3H-fucose application. Labeling of peridermal cells was weak in the early stages of development and ceased in the period of stratum granulosum formation, indicating a stop of glycoprotein synthesis in this embryonal cell layer before its disappearance.

Trypan blue teratogenesis in the rat: further observations in vitro.

Explanted 9 1/2- and 10 1/2-day rat conceptuses were cultured for 24 or 48 hours and 24 hours respectively in immediately centrifuged, heat-inactivated rat serum to which trypan blue was added at concentration of 150 micrograms/ml or 300 micrograms/ml or 450 micrograms/ml. The embryos were assessed for normal growth and differentiation using the following criteria: heart beat, vitelline circulation, fusion of the allantois with the chorion, normal turning, normally closed neural tube, presence of optic vesicles, presence of forelimb buds, tail, somites, somite number, yolk sac diameter, crown-rump length, and protein content. The results indicated that trypan blue is more teratogenic when 9 1/2-day conceptuses are cultured for the first 24 hours, and that its teratogenicity decreases after 10 1/2 days. Morphological abnormalities produced included neural tube defects, turning defects, and tail defects, the tail abnormalities being manifest as fluid-filled blebs. Ten and one-half-day conceptuses cultured for 24 hours in treated serum produced predominantly tail defects. The conceptuses were cultured at 11 days, when the embryo lies completely within the yolk sac. Culture of such conceptuses in serum containing 450 micrograms/ml trypan blue produced mainly tail defects. Injection of 0.5 microliters of 0.5% dye solution into the yolk sac cavity also produced tail abnormalities; sham treatment, or the injection of sterile water or a nonteratogenic dye, azo blue, did not affect embryonic development.

Development of mouse embryos in vitro: preimplantation to the limb bud stage.

Mouse embryos were grown successfully in vitro from the blastocyst stage to the limb bud stage. Mouse blastocysts grown in vitro for 10 days showed blood circulation in the yilk sac, forelimb buds, and the primordia of liver, pancreas, and lungs. These characteristics are indicative of a developmental stage equivalent to one-half of the total gestation period in utero. Improvements in culture conditions from days 7 to 9 have made it feasible to culture mouse blastocysts beyond the early somite stage.