The teratogenic effects of 6-mercaptopurine on chick embryos in ovo.

Fertilized eggs of single-combed White Leghorn hens were each injected through a shell window directly beneath the embryo at 70 or 96 h of incubation with 2 mg of the sodium salt of 6-MP dissolved in 0.1 M phosphate buffer, and at 11 days of incubation the embryos were examined for gross morphological abnormalities. Various gross malformations and growth retardation were found, the most frequently and severely affected structures being limbs, beak, and eyes. Treatment at 70 h caused more severe abnormalities than at 96 h, but the spectrum of defects was not very different.     

Effect of 6-aminonicotinamide in teratogenic doses on the DNA- and RNA-concentration in rabbit embryos.

Pregnant rabbits were injected ip with 5 mg/kg 6-ANA at day 10, 11, 12 or 12.5 respectively of gestation. Embryos removed at day 13 of gestation had reduced DNA and RNA concentrations. The differences (DNA: 14 percent; RNA: 22.4 percent) were significant in the group treated on the twelfth day. Some possible mechanisms are discussed.     

The mechanism of cellular orientation during early cartilage formation in the chick limb and regenerating amphibian limb

A characteristic feature of early cartilage formation is that as the cells at the centre differentiate into chondroblasts they become flattened at right angles to the long axis of the cell mass, whereas the cells in the region of the future perichondrium become flattened circumferentially. On the basis of EM observation a simple model is proposed which is based on matrix secretion flattening the cells.     

The site and sequence of action of 6-aminonicotinamide in causing bone malformations of embryonic chick limb and its relationship to normal development

The results reported here include the following findings: (1) 6-aminonicotinamide (6AN) affects cartilage-producing cells, resulting in subsequent bone malformations. (2) 6AN causes chondrogenic cells to degenerate, a process morphologically similar to that previously described for the action of 6AN on chick limb mesodermal cells in vitro. (3) 6AN acts on the process of phenotypic expression or the newly determined chondrocyte as opposed to acting on mature chondrocytes. Degenerative effects in the limb are not observed until stage 25 corresponding to the first appearance of stainable cartilaginous matrix material. Also, 6AN acts on older embryos to produce zones of degeneration of cartilage involving a process, again, morphologically similar to that described for the action of 6AN on chick limb mesodermal cells in vitro, and again this process does not seem to affect mature chondrocytes. (4) 6AN is effective for a relatively short period, about 36 hr after its introduction into the egg, as shown by the inability of nicotinamide to completely counteract 6AN-caused effects. These observations and others are used to discuss the mode of action of nicotinamide-sensitive teratogens as well as the role of nicotinamide in influencing the expression of limb mesodermal cells into myogenic and chondrogenic phenotypes.     

Time of onset and selective response of chondrogenic core of 5-day chick limb after treatment with 6-aminonicotinamide.

Exposure of the chick embryo to the nicotinamide analog, 6-aminonicotinamide (6-AN), causes specific changes in chondrogenic cells that result in limb deformity. Autoradiography has further delineated these changes and relates them to altered utilization of molecular precursors of cartilage matrix and DNA. With ³⁵SO₄ to monitor synthesis of glycosaminoglycan, it was shown that, at 6 hr and persisting until 24 hr after treatment, 6-AN inhibited utilization of sulfate by cells in the chondrogenic core while having no detectable effect on cells in the chondrogenic periphery. Similarly, 6-AN suppressed incorporation of [³H]thymidine into core cells while having no effect to a slight enhancement effect on chondrogenic and nonchondrogenic cells surrounding the core. These observations support the view that, in response to 6-AN-inhibited NAD(P)-dependent reactions, limb chondrogenic cells (CORE) cease to produce matrix glycosaminoglycan, cease to synthesize DNA, and ultimately succumb. Conversely, presumably as a result of more efficient energy production because they lie closer to a vascular supply of oxygen, cells in the chondrogenic periphery withstand the teratogenic insult and continue proliferating to become the source where subsequent partial repair of the limb occurs.     

The formation of multivesicular structures in the adipose cells of chick embryos

Observation of the cytogenesis of adipose tissue of the chick embryo revealed a quantity of multiversicular structures (MVs) which were found in the intercellular space. Some of them were attached to the adipocytes and others were independently located in the intercellular space. The origin of those MVs appeared to be part of the degenerating mitochondria. Centrally located vesicles and vacuoles in degenerating mitochondria formed a group of short tubules and vacuoles which protruded through the cytoplasmic membrane or bulged out at the edge of the cytoplasmic process. The MVs then spread over the cytoplasmic membrane and finally were discharged from the cell surface as in the manner of apocrine secretion. An invisible barrier between the mass of vesicles and the rest of the cytoplasmic structures appeared to segregate the extruding MVs from the intercellular components such as ribosomes, microtubules, and microfilaments.     

Isolation and characterization of proteoglycans from chick limb bud chondrocytes grown in vitro.

Chick limb bud mesenchyme cells from stage 23-24 embryos were isolated and grown in culture under conditions facilitating chondrogenic development. Dissociative extraction methods were used to isolate proteoglycans from Day 8 cultures, at which time the incorporation of [35S]sulfate into these macromolecules occurred at maximal rates. The monomer (D1) fraction contained 85 to 90% of the proteoglycans originally present in the matrix of the cultures. The composition of this fraction was approximately 7 to 8% protein, 7% keratan sulfate, and 85% chondroitin sulfate. The proportions of nonsulfated, 4-sulfated, and 6-sulfated disaccharides in chondroitinase digests were about 11%, 31%, and 58%, respectively. The D1 fraction exhibited a single, polydisperse component on Sepharose 2B chromatography and in the ultracentrifuge (so = 19 S). In associative density gradients about 35% of the proteoglycans were recovered in a gel at the top of the gradient. The remainder were recovered at the bottom of the gradient in the aggregate (A1) fraction. The A1 fraction exhibited two components, aggregate (about 70% of the total) and monomer, upon Sepharose 2B chromatography and in the ultracentrifuge (so = 120 S for aggregate; 18 S for monomer). The aggregate preparation contained only one of the two link proteins (molecular weight of about 45,000) which occur in proteoglycan preparations from many hyaline cartilages.     

Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.