Spontaneous and retinoic acid-induced postaxial polydactyly in mice

A spontaneous postaxial polydactyly, similar to type B in humans, was found in a partially inbred ICR mouse strain. The supernumerary digit could be detected grossly as early as day 14 of gestation. The incidence of polydactyly decreased with increasing gestational age. All-trans retinoic acid (RA) administered on day 10 or 11 of gestation, but not on day 9, increased the incidence of polydactyly at each gestational day examined. The day 18 levels of polydactyly were greatest after day 10 treatment. No clear dose-response relationship was observed in term fetuses following treatment on day 9, 10, or 11. RA administered on day 10 produced extra digits which were morphologically more advanced than those in the untreated controls. RA, given to the inbred C57B1/10 strain, produced low levels of polydactyly if administered on day 9, but not on day 10 or 11. F1 embryos, from reciprocal crosses between the two strains, were intermediate in response to RA. On day 14, cell death in the postaxial marginal mesoderm was apparent in all protopolydactylous embryos examined, whether treated or untreated. The supernumerary digits varied in size on day 14. The smaller digits appeared to be filled with necrotic mesodermal cells, whereas the larger digits had a necrosis-free area. The size of the extra digit on day 14 seemed to be the most important factor in the persistence of the digit until day 18.     

Disrupting the establishment of polarizing activity by teratogen exposure.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.     

Cadmium induced limb defects in mice: strain associated differences in sensitivity.

Cadmium (CdSO4) was given ip on day 9 at 12 or 24 mumol/kg to pregnant CD-1 (non-inbred) mice. Fetuses showed malformations of the limbs, face, trunk, and tail. There was a statistically significant relationship between the dose of cadmium and the malformation rate. Cadmium (12 mumol/kg ip on day 9) was then given to mice of six inbred strains three of which (A/J, BALB/cJ, and C57BL6J) carry a gene cdm for resistance to cadmium-induced testicular damage, and three strains (AKR/J, CBA/J, and DBA/2J) which do not. Paradoxically, the three strains resistant to cadmium induced testicular damage were significantly more sensitive to its teratogenic effects than were the other three strains. In all inbred strains most malformations involved the limbs. All forelimb defects found in inbred or non-inbred cadmium treated mice were postaxial and indistinguishable from those produced by acetazolamide in mice. The remarkable similarity of the cadmium- and acetazolamide-induced forelimb malformations may be a reflection of the limited number of ways that a rodent forelimb can react to a teratogenic insult. The hindlimb defects were all preaxial.     

Normal development of the skeleton in chick limb buds devoid of dorsal ectoderm

It has been suggested that the ectoderm on the dorsal and ventral faces of the limb bud plays a part in controlling the pattern of cartilage differentiation. To test this, the dorsal wing bud ectoderm in the chick embryo was destroyed by irradiation with ultraviolet light at stage 17-19, at the very beginning of limb bud development, but the apical ectodermal ridge was spared. The irradiated ectoderm disappeared within 24 hr (by stage 23-24) and did not regenerate thereafter; thus the dorsal surface of the limb bud was kept denuded throughout most of the period of skeletal pattern formation. By 6 or 7 days after the irradiation (stage 35), when the rudiments of all the adult skeletal elements are normally present in recognizable form, the irradiated wings could be placed into two categories, those that were approximately normal in shape and those that had curled dorsally. All of these limbs were reduced in size, to varying degrees, when compared to their controls and lacked dorsal soft tissues. The limbs that were normal in shape, however, even though sometimes denuded over practically the whole extent of their dorsal surface, almost always had a complete and normally proportioned cartilage pattern, suggesting that ectoderm (other than the apical ectodermal ridge) does not exert any direct control over the development of the limb cartilage pattern. However, many of those limbs that had curled as a result of the irradiation did have major pattern deformities, suggesting that the topology of cartilage differentiation does depend on the shape of the limb bud.     

Prevention of polydactyly manifestation in Polydactyly Nagoya (Pdn) mice by administration of cytosine arabinoside during pregnancy

Male mice heterozygous for the dominant polydactyly gene Pdn (Polydactyly Nagoya) were crossed with normal or heterozygous females of the same strain. Pregnant females were treated with 5 mg/kg of cytosine arabinoside (Ara-C) on day 12 of gestation. The offspring were removed on day 18 of gestation and examined for external malformations, and the fore- and hindlimbs were examined by means of bone- and cartilage-stained cleared specimens. In +/+ x Pdn/+ matings, Pdn/+ fetuses, bearing preaxial polydactyly of the distal phalangeal type in the hindlimb and deformity of the 1st digit in the forelimb, were obtained in about 50% of the nontreated group. In treated fetuses, however, the incidence of polydactyly and deformity of the 1st digit decreased to 1.4 and 10.1%, respectively. Nontreated Pdn/Pdn fetuses exhibited preaxial polydactyly of the duplicated or triplicated metacarpal/metatarsal type both in the fore- and hindlimbs. In the treated Pdn/Pdn fetuses, the number of preaxial extra digits decreased in both limbs. Some hindlimbs of the treated Pdn/Pdn fetuses exhibited five metatarsals, normally. In the vitally stained specimens at 6 and 24 hours after injection of Ara-C, preaxial marginal necrotic zones (fMI) were observed in almost all of the treated embryos from +/+ x Pdn/+ matings. However, approximately half of the embryos did not exhibit fMI in the nontreated control group at the same stage. Those embryos deficient in fMI were regarded as Pdn/+. These findings indicated that a subteratogenic dose of Ara-C prevented the genetic expression of polydactyly in almost all Pdn/+ and some cases of Pdn/Pdn mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin teratogenesis: defects produced by concanavalin A in fetal rabbits.

Concanavalin A (con A) is teratogenic to rabbit embryos during gestational days 12--15. Intracoelomic injections of 40 microliter con A solution (4 microgram/microliter) were performed on rabbit embryos during gestational days 10--15. Control embryos received either 40 microliter of saline, sham injection or no treatment. Con A caused increased fetal resorptions on days 10 and 11, but malformation levels did not differ from controls. On days 12--15, con A produced craniofacial, trunk and limb anomalies. The highest percentage of malformation occurred on day 14. The defects were classified into four groups: (1) malformations of limbs including paw and digital dysplasias as well as fusions of the limbs to the head or body wall; (2) "closure" defects such as umbilical hernia, encephalocoele, exencephaly or ectopia cordis; (3) "contracture" defects such as club paws, extended knees, or clenched digits, which exhibited normal osseous and cartilaginous skeletons; and (4) miscellaneous, non-specific anomalies including fused or dysplastic sternebrae or ribs. Histologic analysis of selected 12-day embryos 4 to 18 hours post-injection was performed to ascertain potential sites of teratogenic action. At 12 hours ectodermal necrosis was observed in the limb buds adjacent to the apical ectodermal ridge. By 18 hours, the ectoderm had eroded, exposing the basal lamina to the amniotic fluid. Focal areas of mesenchymal necrosis were observed in association with the ectodermal erosion. The potential roles of amniocentesis and limb bud repair in the genesis of the malformations are discussed.     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

Teratogenic effects of chlorambucil on in vivo and in vitro organogenesis in mice.

Pregnant ICR/DUB mice were each given a single oral injection of chlorambucil (14.2 or 20 mg/kg) on the 10th, 11th, 12th, or 13th day of gestation (plug day = 1st day). Fetuses examined on the 18th day were decreased in weight and had tail, cranial, and limb defects. They type and frequency of malformations differed according to the dosage and day of treatment. Limb defects resulted from treatment on the 11th or 12th days of gestation and tail defects from treatment on all days. Control limb buds from 12th day embryos cultured for 6 days in serum-supplemented BGJ medium containing 0.5-2 mug/ml chlorambucil were retarded in development and had cartilage abnormalities. The extent of the deformities was dose related. Limb buds were also taken from embryos 24 h after in vivo exposure to teratogenic doses of chlorambucil and cultured in control medium. After 6 days in culture these limbs also had growth impairment and cartilage abnormalities. The defects in limbs exposed in vitro were similar to those in limbs exposed in vivo.     

Retinoic-acid-induced limb-reduction defects: perturbation of zones of programmed cell death as a pathogenetic mechanism

Pregnant C57Bl/6J mice were treated with 100 mg/kg body weight of all-trans retinoic acid in sesame oil on day 11.0 of gestation. Among the live fetuses harvested on day 18 of gestation, 100% had mesomelic defects of the limbs as determined by gross examination and skeletal staining. Control fetuses treated with sesame oil had no observable limb malformations. Some treated and control embryos were harvested 12 hr after treatment and examined for patterns of cell death by using the supravital stain Nile blue sulphate and methylene-blue- and acid-fuchsin-stained histological sections. Retinoic-acid-induced cell death in the core of the limb was always associated with the zones of programmed cell death as seen in control embryos of comparable stages. This, in concert with previous studies demonstrating excessive cell death in regions of programmed cell death that correlated with subsequent malformations, leads us to conclude that the pathogenesis of mesomelic malformations has a primary association with the phenomenon of programmed cell death.     

In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb

To determine the role of Bone morphogenetic protein (BMP) signaling in murine limb development in vivo, the keratin 14 promoter was used to drive expression of the BMP antagonist Noggin in transgenic mice. Phosphorylation and nuclear translocation of Smad1/5 were dramatically reduced in limbs of the transgenic animals, confirming the inhibition of BMP signaling. These mice developed extensive limb soft tissue syndactyly and postaxial polydactyly. Apoptosis in the developing limb necrotic zones was reduced with incomplete regression of the interdigital tissue. The postaxial extra digit is also consistent with a role for BMPs in regulating apoptosis. Furthermore, there was persistent expression of Fgf8, suggesting a delay in the regression of the AER. However, Msx1 and Msx2 expression was unchanged in these transgenic mice, implying that induction of these genes is not essential for mediating BMP-induced interdigital apoptosis in mice. These abnormalities were rescued by coexpressing BMP4 under the same promoter in double transgenic mice, suggesting that the limb abnormalities are a direct effect of inhibiting BMP signaling.     

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.     

Changes of the cell surface charge of amphibian ectoderm after induction

Summary Isolated ectoderm of early gastrula stages ofTriturus alpestris was treated with vegetalizing factor for 24 h employing the sandwich method (induced ectoderm). Controls were incubated for the same period with -globulin which has no inducing activity. Explants of both series were labelled with cationized ferritin, which binds to negatively charged groups at physiological pH. In non-induced ectoderm, ferritin particles can be found as a thin layer all over the plasma membranes. In induced ectoderm the total amount of ferritin bound to the plasma membrane is much lower than in non-induced ectoderm. Ferritin is located in restricted areas only. In contrast to the controls, other membrane areas are free of ferritin particles. The correlation between these results and the change of cell affinity after induction with vegetalizing factor is discussed.     

A new interpretation of the necrotic changes occurring in the developing limb bud paddle of mouse embryos based upon recent observations in four different phenotypes.

Degenerative changes occurring in the apical ectodermal ridge (a.e.r.) and undifferentiated distal mesoderm of developing limb buds were studied macro- and microscopically in day-11 to day-13 mouse embryos displaying the normal (+/+), oligosyndactylous (Os/+), polydactylous (Xpl/+) and hybrid (Os/+/Xpl/+) phenotypes. Isolated limb buds were submitted either to supravital staining with Nile blue sulfate or to lectin binding staining in serial paraffin sections, taking advantage of strong binding affinites of macrophage cells for peanut agglutinin after neuraminidase treatment and for ricinus communis agglutinin. Necrotic changes detected in three definite areas of the distal mesoderm of normal limb buds exhibit characteristic spatial temporal relationships with earlier cytolytic changes affecting the pre- and postaxial parts of the a.e.r. Two of them, known as the primary preaxial site (fpp) and the anterior marginal necrotic zone (AMNZ) appeared deeply modified in mutant embryos as compared to the posterior marginal necrotic zone (PMNZ) which remained unaffected. Macrophage cells loaded with cell debris appear in advance and in excessive number in the fpp of Os/+ limb buds. Conversely, they were found absent or locally reduced in number in the fpp and AMNZ of Xpl/+ limb buds which otherwise develop in the same area a preaxial protrusion covered with a healthy portion of the a.e.r. Hybrid Os/+/Xpl/+ limb buds expressing both mutant genes develop a smaller and macrophage-free preaxial protrusion which coexists with residual and locally excessive necrotic changes in its immediate surrounding and is covered with a normally necrotic portion of the a.e.r. Microscopic observations collected in the limb buds of all phenotypes, though more frequently in Os/+ limb buds, strongly suggest that in all three necrotic sites examined, macrophage cells of vascular origin somehow contribute to the clearance of ectodermal necrotic debris and eventually return in the blood stream through the marginal vein and its affluents.     

Cyclosporine A dosing-time-dependent effects on plasma creatinine and body weight in male Wistar rats treated for 3 weeks.

Cyclosporine A (CsA) nephrotoxicity was assessed in 120 male Wistar rats (350 +/- 50 g) entrained to a 12-h cycle (light-dark 12:12); plasma creatinine level and body weight were examined in controls and in rats that had been treated daily with oral CsA or vehicle alone (olive oil-ethanol 90:10) for 21 days; daily dosing (40 mg/kg) was at one of six equally spaced given times during the 24-h cycle. The variations observed in both indexes were shown to be circadian dosing stage dependent. Nephrotoxicity was present as early as the third day of treatment with CsA; plasma creatinine level was enhanced by about 50% in rats dosed around the time of the change from darkness to light: at 22 HALO, 146.7 +/- 4.5 mumol/L, against 92.0 +/- 2.8 mumol/L for controls (p less than 0.05); and at 2 HALO, 148.3 +/- 10.0 mumol/L, against 95.0 +/- 4.3 mumol/L for controls (p less than 0.05). Thereafter, a remission episode was observed between days D5-D9. The more drastic effects were seen on days D16 and D21, in animals dosed in the beginning of the dark span (14 HALO): 185 +/- 10 mumol/L for CsA and 98.0 +/- 5.3 mumol/L for controls (p less than 0.01) and, to a lesser extent, in rats treated at the early resting phase (2 HALO): 152.4 +/- 31 mumol/L for CsA and 95.0 +/- 4 mumol/L for controls (p less than 0.05). The normal increase in body weight during the 21-day period (about 14 +/- 8% in controls) was impeded in CsA-administered rats, especially those dosed at the beginning of the activity span (14 HALO) that even suffered weight reduction. Differences in percentages of survivors were noticed, depending on dosing stage. About 40% of the animals in every time CsA-treatment group died, except for those dosed at the end of the resting period (10 HALO), when all animals died. In surviving rats, the cessation of CsA dosing resulted in a reversible effect on the study variables.     

Effects of orthodontic tooth movement on the Alcian blue staining patterns of rat alveolar bone: an histochemical study

There is little information available concerning the effects of orthodontic forces on glycosaminoglycans (GAG) of alveolar bone. The present study identifies changes in Alcian blue staining intensity in rat alveolar bone undergoing resorption resulting from a heavy (25g) tipping force applied to the adjacent teeth by a separating spring. One day after force application, bone from treated animals (internal control and experimental sides) demonstrated more intense staining with Alcian blue, pH 1.0 (p less than 0.005) and pH 2.5 (p less than 0.05) than external controls (untreated animals). By day 3, the intensity of Alcian blue staining of treated alveolar bone was similar to untreated. Chondroitinase AC, ABC and testicular hyaluronidase predigestion did not completely block the staining reaction, suggesting that both GAG and noncollagenous proteins were demonstrated. Mean cross-sectional areas of the interdental septum of the experimental side were nearly 44% less than that of the internal control side after 3 days and nearly 62% less after 5 days. The study suggested that alterations in bone GAG levels occurred prior to tooth movement as histochemical changes occurred after force application but before initiation of significant septal resorption. A precise appraisal of the types of macromolecules effected awaits future biochemical analysis. The results of the present work strongly suggest the use of an external control group for future studies, as Alcian blue staining reactions of the internal control side of treated animals were not similar to those of external controls.     

Histological study of cell death in digital malformations induced by 5-azacytidine: suppressive effect of caffeine

The present study investigated microscopically the process of 5-azacytidine (5-AC)-induced digital teratogenesis and caffeine's suppressive effect on this process. Three distinct zones of programmed cell death were observed in control and caffeine-treated embryos 3 hours after 5-AC injection: the preaxial and postaxial ectodermal regions and the central part of the mesodermal regions. 5-AC temporarily suppressed programmed cell death in the ectoderm and mesoderm 3 hours after it was injected. However, caffeine promoted programmed cell death; normal programmed cell death was observed in the limb buds of embryos whose dams were treated with 5-AC and caffeine. The percentage of total cell death in hindlimb buds of embryos treated with 5-AC and caffeine was higher than that from embryos treated with 5-AC, whereas 5-AC-induced digital malformations were reduced by post-treatment with caffeine. Cell death reached a maximum 12 hours after the injection in limb buds from 5-AC and caffeine-treated embryos and at 24 hours in the 5-AC treated embryos. Furthermore, in the 5-AC and caffeine-treated embryos, the frequency of cell deaths at 12 hours increased almost linearly with the doses of caffeine in parallel with the reduction of 5-AC-induced malformation frequency by caffeine. These results suggest that although induced cell death may be one of the factors leading to digital malformations produced by 5-AC, it is not essential, and the existence of other factors affecting the pattern formation of the limb bud is proposed.     

Inhibitory effect on limb morphogenesis by cells of the polarizing zone coaggregated with pre- or postaxial wing bud mesoderm.

The influence of cells of the polarizing zone mesoderm on the morphogenesis of recombinant chick limbs was studied. The recombinant buds were composed of leg bud ectoderm and different regions of the wing bud mesoderm, which had been dissociated and reaggregated. In any case where the polarizing zone mesoderm was coaggregated with the wing mesoderm the morphogenetic capabilities of the recombinant were reduced. This was the case with postaxial mesoderm, preaxial mesoderm plus polarizing tissue, and postaxial mesoderm from which a piece of the nonpolarizing mesoderm (comparable in size to the polarizing zone) had been removed. All of these gave outgrowths with digits in only a very low percentage of cases. In contrast, those recombinants without polarizing mesoderm developed outgrowths with digits in a high percentage of cases, indicating good morphogenesis. Finally, if the polarizing zone were removed prior to dissociation, the recombinant limb, composed of the total remaining wing bud mesoderm plus leg bud ectoderm, exhibited a higher percentage of complete morphogenesis than if the polarizing zone had been part of the recombinant.It is clear that cells of the polarizing zone, when dissociated, and coaggregated with wing mesoderm, are inhibitory to the morphogenetic performance of that mesoderm in the recombinant limb situation.     

The effects of minoxidil on the development of the chick embryo

The effects of minoxidil were studied on chick embryos of 24 and 48 hours of incubation. Minoxidil (3%) was injected into the air sacs of the eggs at doses of 20, 30, 40, and 50 microliters per egg. The controls received 100 microliters of physiological saline. All the embryos, including controls, were examined at day 13. The total number of eggs used in this study was 300. At 24 hours incubation, the percentage of survival ranged from 87 to 21 as the dosages of minoxidil were increased from 20 microliters to 50 microliters per egg (controls = 87%). The survival of the embryos ranged from 79% to 9% after the 48-hour treatment with the similar dosages of minoxidil utilized for the 24-hour group (controls = 83%). A low incidence of gross malformations such as twisted limbs, abnormal beak, short neck and everted viscera were observed; however, the increased incidence was not statistically significant when compared to controls. Body hemorrhage and edema were of high occurrence among the treated embryos. These effects are probably secondary to the known pharmacological effects of minoxidil. The frequency and types of gross malformations did not vary much in the 24 or 48-hour treated groups.     

Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.     

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.