Morphological analysis of abnormal digital chondrogenesis in the brachypod (bpH) mouse limb in organ culture.

Brachypod (bpH/bpH), an autosomal mutation in mice, is characterized by a shortening of the long bones and paws, and a delay or absence of ossification in some of the distal limb elements. The present study represents a detailed description of the brachypod phenotype in day 12 hindlimb buds maintained for 6 days in a submerged, serum-free organ culture system. Using this in vitro system, the proximal-to-distal effect on the severity of cartilage reduction was intensified in the brachypod explants with an intermediate expression in the heterozygotes. Immunofluorescent staining of the brachypod cartilage revealed a deficiency in and an abnormal distribution of the proteoglycans. Although there was no recognizable difference in the immunofluorescent staining for type II collagen between the mutant and wild-type, electron micrographs showed the presence of thick fibrils in the matrix. Other atypical structures in the brachypod cartilage included pleomorphic nuclei, reduced intracellular glycogen granules and profuse intercellular contacts. It is proposed that with the use of this in vitro system which supports the autonomous development of the individual limb elements, experiments concerning the pathogenesis of skeletal mutations such as brachypod should be more feasible.

Effects of isotretinoin (13-cis-retinoic acid) on the development of mouse limbs in vivo and in vitro.

Isotretinoin (13-cis-RA) is known to be teratogenic in humans and laboratory animals. The relatively low potency of 13-cis-RA in NRMI mice in comparison to the all-trans isomer has been proposed to be due to minimal transfer across the placenta (Creech-Kraft et al., '87). To further delineate the teratogenic potential of 13-cis-RA, a dose-response, temporal study was conducted in vivo and in vitro using submerged limb culture and image analysis evaluation of development. Dose-dependent embryotoxicity was produced by treatment on GD 7, while later treatments produced inconsistent effects on resorption rate and fetal weight. Treatment on either GD 7 or GD 8 produced a number of malformations in dose-dependent manner. Most common were tail and cleft palate defects, which were produced by 13-cis-RA on each of the days tested (GD 7-GD 11), with peak malformations occurring on GD 9 and GD 10 for tail and cleft palate, respectively. Most limb defects were produced after GD 10 and GD 11 exposure. The observed frequency of defects confirmed that in ICR mice 13-cis-RA is about 10-fold less potent than all-trans-RA as a limb teratogen (Kwasigroch and Kochhar, '80; Kochhar and Penner, '87). Effects observed via image analysis following maintenance of limbs in serum-free culture medium were dose dependent. Low dose treatment produced occasional polydactyly. The intermediate dose caused somewhat variable region-dependent increases in cartilaginous bone anlagen area. The high dose of 13-cis-RA produced irregular limb outlines, a reduction in bone anlagen area, and an inhibition of alcian blue staining of cartilage without affecting morphogenesis of bone anlagen. These results confirm that, when the effects of the administered doses are evaluated, 13-cis-RA is a much less potent teratogen in comparison to the all-trans isomer. More importantly, the results show that retinoids can enhance (at low and intermediate doses), depress (at high doses), or eliminate (high dose) chondrogenenic expression during limb morphogenesis in vitro. This indicates that retinoids such as 13-cis-RA can manipulate events in development in a variety of ways (i.e., produce malformations, interfere with chondrogenic expression without affecting morphogenesis, and stimulate growth) in a dose- and time-dependent manner. Although the ability of RA to act as a true morphogen has recently been questioned (Wanek et al., '91; Noji et al., '91), the results presented here support the position that RA can modulate the development of the limb (and probably other organ systems) in several vertebrate species.

Alterations in cell surface galactosyltransferase activity during limb chondrogenesis in brachypod mutant mouse embryos.

The autosomal mutation brachypod (bpH/bpH) in the mouse affects the development of precartilage mesenchymal condensation in the limb-bud. We have previously shown that this defect is localized to the expression of terminal N-acetylglucosamine (GlcNAc) glycoproteins in the plasma membrane (Elmer and Wright, '83). The present study is focused on cell surface galactosyltransferase (GalTase), an ectoenzyme that transfers galactose to its GlcNAc substrate. Purified plasma membrane preparations derived from wild-type (+/+), heterozygote (+/bpH) and brachypod (bpH/bpH) embryonic mouse limb cells were assayed for GalTase activity during in vitro and in utero chondrogenesis using High-Performance Liquid Chromatography (HPLC). On embryonic day E12, prior to overt expression of the mutant gene, no significant difference in GalTase activity was observed. By the third day in culture, all major chondrogenic elements of the autopod were present in +/+ and +/bpH embryos, whereas the mutant autopods were markedly deficient in staining and appeared consistently shorter. The accumulation of alcianophilic cartilage matrix in the wild-type was accompanied by a 29% increase in GalTase activity, which reflected the net change (29%) observed during development from days E12 to E13 in utero. The GalTase activity for the in utero E13 mutant (13%) was significantly different from control. In culture, day E12 mutant autopods actually decreased in their GalTase level by 3 days so that the activity was reduced to only 57% of the wild-type. Though GalTase activity in the heterozygote showed an intermediate expression, optical image analysis did not reveal consistent differences in cartilage development when compared to +/+, arguing against a gene-dosage effect at the gross anatomical level. These data indicate that an increase in plasma membrane GalTase activity is a natural developmental event that occurs during limb-bud chondrogenesis and a decrease in GalTase activity contributes to the dysmorphogenesis in brachypod limb-buds.

Retinoic acid enhances and depresses in vitro development of cartilaginous bone anlagen in embryonic mouse limbs.

Forelimbs of Day 11 and Day 12 embryonic mice were excised and cultured for 3 d in the presence of either 0.25 microgram (8 X 10(-7) M), 0.5 microgram (1.7 X 10(-6) M), or 1.0 microgram (3.3 X 10(-6) M) of all-trans retinoic acid (RA) per milliliter of culture medium. Cultured limbs were fixed, stained, and mounted whole on glass slides and evaluated with computerized optical image analysis for RA-induced effects on the area and shape of the total limb and individual bone anlagen. Relative effects of RA on total bone, soft tissue, long bone, and paw regions were also examined. With Day 11 forelimbs total bone area was increased by 10.5% by the low dose of RA. The increase was mostly in long bones and at the expense of soft tissue. Total bone area was increased 9.3% with Day 12 forelimbs. This increase was primarily in the paw. The high dose of RA decreased Day 11 forelimb area, primarily affecting long bones. Day 12 forelimbs were not significantly affected by the high dose of RA. Effects of the intermediate dose were primarily limited to reduction in soft tissue area. Long bone:paw and soft tissue:bone ratios reflected these effects. The high dose produced a consistent rounding or shortening of Day 11 forelimb bones. On Day 12 0.5 microgram/ml RA produced an inconsistent pattern of rounding of bone anlagen. Treatment with the high dose on Day 12 produced angular rather than rounded contours in many cases, as indicated by shape factor values closer to zero than obtained with controls. These data show that direct exposure to RA can affect both the size and shape of bone anlagen of the developing limb; the low dose enhances and the high dose depresses development. The results support previous studies which suggest that RA may play a critical role in the control of cell activities such as cell migration, proliferation, and cytodifferentiation in the development of the cartilaginous bone anlagen.

The teratogenic interaction of hydroxyurea and 5-bromodeoxyuridine examined with the aid of limb culture and image analysis.

Pregnant mice were treated on gestation Day 11 (E11) with a single dose of either hydroxyurea (HU, 250 mg/kg) or 5-bromo-2'deoxyuridine (BrdU, 500 mg/kg), or with a combination of the two agents. The dams were either allowed to go to term, when the fetuses were examined for cleft palate (CP) and digital anomalies, or killed 24 hr after the treatment. In the latter case, limbs from the embryos of control and treated dams were excised and cultured for 6 days in submerged culture. At the end of the culture period, the limb explants were stained for cartilage. The various cartilaginous components were subsequently analyzed using image analysis methods. CP was observed in 2.4 and 22.9% of the fetuses, respectively, after a single dose of HU or BrdU on E11. Simultaneous HU + BrdU treatment decreased the incidence of BrdU-induced CP to 3.6%, while treatment with BrdU 3 hr after HU resulted in 33.3% CP. In addition, syndactyly and ectrodactyly, not seen after the treatment with either HU or BrdU alone, were observed when the two agents were administered simultaneously, 1 hr apart or with a 3-hr delay. The teratogenic response was enhanced when limbs were cultured for 6 days. Digital anomalies were observed in the limb explants of BrdU-pretreated embryos; such abnormalities were not observed in vivo. Image analysis of cultured limb explants revealed that with HU-3 hr-BrdU pretreatment, the total limb area occupied by the long bones was increased at the cost of the paw area. However, no consistent changes in the shape or form of individual bones were observed. HU and BrdU given together produced a teratogenic response which was significantly different from that observed following the administration of HU or BrdU alone. This is considered to be due to an interference with the incorporation of BrdU in the DNA, since HU is known to block DNA synthesis. When HU was given before BrdU, the effects of BrdU were enhanced, probably due to the reported synchronizing action of HU on dividing cells. We also found that after a similar in utero exposure, the culture conditions themselves enhanced the frequency of teratogenic expression of BrdU.

Evaluation of simultaneous teaching of extremities in gross anatomy program.

A gross anatomy program was designed to expose medical students to all areas of the body but shortened the dissection time on the extremities by having half the class dissect either the upper or lower extremity and then study the opposite extremity already dissected by other classmates. The program has been used for six years and was evaluated via an analysis of covariance by comparing the intramural examination performance on both the dissected and undissected extremities. There was no statistical difference in the students' performances regardless of the extremity dissected. The program was also evaluated externally by student performance on Part I of the examinations of the National Board of Medical Examiners. Students (n = 191) performed at the national average (69 percent) on all gross anatomy questions and on those questions pertaining to either extremity (66 percent). The program has efficiently utilized laboratory time with no measurable change in performance by six medical classes.

Estimation of creatine kinase isoenzymes: the effects of caffeine and retinoic acid on CK levels in fetal mouse limbs.

Caffeine and retinoic acid were examined for effects upon limb morphogenesis and upon creatine kinase (CK) as a measure of limb myogenesis. Caffeine at 200 mg/kg, i.p., on E11 produced a low level of forelimb (1.2%) and hindlimb (2.0%) defects. Retinoic acid, at 50 mg/kg given orally as an oily suspension, induced a high level of reduction deformities. Hindlimbs (100%) were affected more than forelimbs (88%). Limbs (E16) were examined for CK isoenzymes using DEAE-Sephacel column chromatography. Untreated limbs had 88.04% skeletal muscle (MM), 6.98% hybrid (MB) and 5.08% brain (BB) CK isoenzyme. Caffeine had no effect. However, retinoic acid increased MM-CK to 92.67%, and decreased BB-CK to 2.24%. This is the first evidence that suggests that retinoic acid may modify the phenotypic expression of developing muscle.

The toxicology of chemical interactions during pregnancy in the mouse: caffeine and phenytoin.

The toxic interaction of caffeine and phenytoin during pregnancy was investigated in mice of the ICR strain on E10 of gestation. Caffeine, over a range of dosages, showed limited embryotoxic activity. Phenytoin was also weakly teratogenic and dosages needed to elicit embryotoxicity were accompanied by a significant increase in maternal lethality. Pretreatment with caffeine enhanced phenytoin-induced toxicity and teratogenicity and these observations confirm that caffeine has the ability to function as a co-teratogen. Pretreatment with phenytoin produced a significant increase in maternal lethality following caffeine administration but no co-teratogenic effect. It is suggested that these results are the consequence of a yet undefined interaction at critical receptor sites in the maternal-embryo unit.

Mouse limb bud development in submerged culture: quantitative assessment of the effects of in vivo exposure to retinoic acid.

Retinoic acid, suspended in cottonseed oil, was administered via gavage to pregnant mice (ICR strain) on day 11 (E 11) of gestation at doses of either 20, 40, or 80 mg/kg. Fetuses were examined for external malformations on day 17 (E 17). Retinoic acid treatment induced micromelia (with the elimination of several long bones at higher doses) and digital defects (ectrodactyly and syndactyly) in a dose-dependent manner in fetuses examined on day 17. Hindlimbs were affected more than forelimbs. In another group of experiments, limbs exposed to retinoic acid treatment in utero on E 11 were cultured on E 12 and maintained for 3 days in submerged culture. Cultured limbs were examined qualitatively for digital and long bone defects, and image analysis of the area and form of bone anlagen of cultured limbs was used to quantitatively evaluate the teratogenic potential of retinoic acid. The qualitative evaluation indicated that the retinoic acid-induced effects obtained in vivo and with pretreated, cultured limbs were essentially the same, except that the severity of regional effects changed as a result of culture. The incidence of ectrodactyly was higher with cultured limbs than with E 17 fetal limbs, but fewer cultured limbs were missing long bones. These results suggest that culturing limbs, after they have been pretreated in utero, modifies their response to a teratogen and demonstrates that the paw skeleton is extremely sensitive to teratogen treatment under these experimental conditions. Therefore, care must be exercised when attempting to compare in vivo and in vitro teratogenic data. This study also clearly demonstrates the power and usefulness of image analysis for quantitative evaluation of both the area and form of a cultured specimen such as the developing limb bud. Quantitative, image analysis of cultured limbs showed a dose-dependent decrease in area of both fore- and hindlimbs. The effect was most severe in hindlimbs. In the forelimb, the paw was affected more than the long bones; as the dose increased, this disparity of effect also increased. With the hindlimb, a greater effect on the paw occurred only at 80 mg/kg. Computing the soft tissue/bone ratio illustrated that retinoic acid had a greater effect on chondrogenic tissue than on soft tissue.

Cross-sectional anatomy: preparation of teaching specimens.

Forty cross sections of a male cadaver were prepared for a Continuing Medical Education Seminar and have been used in teaching programs for the past 3 years. The sections are easy and inexpensive to construct and can be maintained with a minimal amount of effort.

The interaction of chemicals during pregnancy: an update.

The role of chemical interactions in the genesis of induced embryotoxicity in humans and in experimental animals is a point of current concern and interest. This review summarizes the current knowledge in this field and demonstrates that, under appropriate conditions, the observed results of chemical interactions can be explained, in part, through an understanding of the underlying pharmacokinetic and pharmacodynamic parameters. These define the concentration of the "primary" teratogen at its receptor site in cells and tissues that are particularly sensitive to its embryotoxic action. The interacting "secondary" or "coteratogen" is viewed as an agent that is capable of altering these parameters in a way that is correlated with an observed alteration in the embryotoxic response. A series of recent experiments (diazouracil: 5-fluouracil; hydroxyurea: 5-bromodeoxyuridine; caffeine: 5-bromodeoxyuridine; caffeine: phenytoin) are reported which demonstrate both the validity and the limitations of such an approach and attempt to interpret observations, which are primarily empirical in nature, in pharmacologic terms.

Vitamin A alters the internal viscosity of fragments of limb-bud mesenchyme.

Two techniques were used to examine the effect of vitamin A compounds (vitamin A acid = retinoic acid and vitamin A acetate) upon the relative strengths of adhesion among mouse limb-bud mesenchymal cells. Treatment with retinoic acid in vivo and with vitamin A acetate in vitro reduced the rate at which the fragments of mesenchyme rounded-up when cultured on a non-adhesive substratum, but these compounds did not alter the behavior of tissues tested in fragment-fusion experiments. These conflicting results indicate that the two tests measure different activities of cells and suggest that treatment with vitamin A alters the property(ies) of cells which regulate the internal viscosity of tissues.

Production of congenital limb defects with retinoic acid: phenomenological evidence of progressive differentiation during limb morphogenesis.

Maternal administration of a single dose of retinoic acid (vitamin A acid, 100 mg/kg) on either the 11th, 11 1/2, 12th, 12 1/2, 13th or 13 1/2 day of gestation produced phocomelia or partial phocomelia in ICR/DUB fetuses. The results depended upon the time of treatment and two gradients of effect were produced: 1) cranio-caudal gradient, since forelimb defects resulted from between days 11 and 13, while similar hindlimb abnormalities were produced by administration of retinoic acid 12 to 24 hours later: 2) proximo-distal gradient, due to the heterogenous sensitivity among individual bones of the limb. In the forelimb, early treatment (11th day) produced humero-unlar defects and later treatment (12th day) ulnoradial defects. A similar proximo-distal gradient was observed in the hindlimb. The use of teratological studies as a tool to assist morphogenetic investigation is discussed.

Estimation of creatine phosphokinase and hydroxyproline in the developing limb: its use in evaluating the effect of teratogens on myogenesis and chondrogenesis.

Forelimbs of mouse fetuses were examined for tissue-specific, drug-induced alterations in their biochemical composition. The activity of the enzyme creatine phospholinase (CPK; to estimate myogenesis) and the content of hydroxyproline (HP; to estimate chondrogenesis) were compared in homogenates of control and treated mouse-fetus forelimbs on day 14 of gestation. In addition, the content of DNA, RNA, and protein was also measured. Injection of 6-aminonicotinamide (6-AN) (15 mg/kg) on day 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a significant reduction in day 14 forelimb HP and RNA content, without altering CPK, DNA, or protein; thus, the HP:CPK ratio was decreased. These results indicated that 1) 6-AN nonspecifically retards growth and cyto-differentiation in limbs; 2) retinoic acid inhibits synthesis of collagen and RNA; 3) retinoic acid has a differential effect upon chondrogenic and myogenic tissues of the limb, and 4) the comparison of HP content and CPK activity in tissue homogenates is an acceptable method of evaluating teratogenic compounds for selective effects.