Apoptosis in the chick wing bud and the permanence of FGF-2 rescue.

Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The opaque patch (OP) and posterior necrotic zone (PNZ) were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by inhibition of protein synthesis with cycloheximide. Both regions showed rescue with cycloheximide as determined by the chromium release assay and examination of electron micrographs. Also, the permanence of basic fibroblast growth factor (EGF-2) rescue in the OP and NPZ was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. This research shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation and is prevented by inhibition of protein synthesis with cycloheximide, biochemically characterizing this death as apoptosis. It also suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ.

The control of cell death in the early chick embryo wing bud.

Developmentally programmed cell death occurs in several regions of the chick wing bud. We have studied the nature and control of this cell death in vitro in tissues from two of these regions, the posterior necrotic zone (PNZ) and the opaque patch (OP). When tissue from these regions is excised prior to normal cell death and placed into organ culture, cell death ensues. Under these conditions, cell death in tissue from both of these regions is inhibited by fibroblast growth factor-2 (FGF-2). The only other growth factor we have found to have this function is insulin-like growth factor-II. Cell death in tissue from the OP and PNZ occurs by apoptosis, as indicated by the internucleosomal degradation of DNA and the inhibition of cell death by cycloheximide, an inhibitor of protein synthesis. If cell death is inhibited by FGF-2 and then the growth factor is washed away, a compensatory burst of cell death occurs in the PNZ tissue but not the OP tissue. This finding may indicate that in the PNZ, a death program progresses in the face of FGF-2 inhibition, resulting in more cells on the brink of death when the growth factor is removed.

Fibroblast growth factor and culture in monolayer rescue mesoderm cells destined to die in the developing avian wing.

In an effort to elucidate control mechanisms for developmentally programmed cell death, conditions were sought that rescue the cells destined to die. Three areas of mesodermal cell death in the chick wing were examined: the posterior necrotic zone (PNZ), the opaque patch (OP), and apical mesoderm. The PNZ and OP are areas of normally programmed cell death, whereas the apical mesoderm undergoes cell death only after the overlying apical ectodermal ridge is excised. Cell death in vitro was quantitated using the chromium-release assay. While these tissues undergo apparently normal cell death in organ culture, in monolayer culture almost all are rescued. In addition, the cells are rescued by the addition of fibroblast growth factor to organ cultures. Since fibroblast growth factor is present in decreasing amounts in the limb at this stage of development, normal cell death may occur upon withdrawal of growth factor.

Studies of methotrexate-induced limb dysplasias utilizing a 51chromium release assay.

The folate antagonist methotrexate (MTX), widely used in chemotherapy, is a well-documented teratogen. However, the mechanism by which it exerts its effects is still unclear. Specifically, we have examined the cytotoxicity of MTX in vivo and in vitro and have looked at the relationship between cytotoxicity and teratogenesis. The chick embryo was utilized to examine the effects of the drug administered to carefully staged embryos. Embryos were exposed at stages 18-22 and examined on day 11 of incubation. Wings were malformed in a stage-dependent manner while legs were affected similarly at each stage used. A modification of the 51chromium-release assay was used to test the toxicity of MTX to limb cells in vitro. None of the tissues tested showed measurable toxicity in vitro even though the drug kills cells in vivo, thereby suggesting that MTX may be metabolized differently in vitro. Malformations induced by MTX do not seem to be due to changes in the amount of cell death taking place in the limb but may be caused by a transient inhibition of cell division.

Transfer of dorsoventral information from mesoderm to ectoderm at the onset of limb development.

Control of dorsoventral patterns in the chick at the prelimb stages resides in the limb mesoderm. Recombination experiments at stage 14, with dorsoventrally reversed ectoderm, result in wings with mesodermal dorsoventral polarity. Similar recombinations at stage 16 show that the ectoderm has acquired dorsoventral information and can impose this polarity on the patterns of mesodermal differentiation in the distal regions of the wing. The dorsoventral information in the ectoderm comes from the mesoderm, which transfers this information to the overlying ectoderm between stages 14 and 16. The initial dorsoventral overlying ectoderm between stages 14 and 16. The initial dorsoventral information in the ectoderm is not stable and can be reprogrammed by stage 14 mesoderm. Subsequently, there is a gradual stabilization of the ectodermal information. At the same time the mesoderm loses its capacity to reprogram dorsoventral information in the ectoderm.

In vitro effects of calmodulin antagonists on macrophage function in the posterior necrotic zone of the chick wing.

Excised tissues from the prospective posterior necrotic zone (pPNZ) of the stage 21 chick wing were cultured in the presence of the calmodulin antagonists/protein kinase C inhibitors trifluoperazine (TFP) or chlorpromazine (CPZ). The appearance of cell death in vitro was not affected by the drugs. Macrophages differentiated normally and were competent to engulf debris. Lysosomal fusion with phagosomes and the digestion of most debris also occurred in the presence of the drugs. However, the macrophages were unable to process internalized cell membranes properly and continued accumulating membrane until they were grossly distended. The effect was reversible upon removal of the drugs. The results suggest a role for calmodulin and/or protein kinase C in the proper recycling of internalized membrane in embryonic macrophages.

Ectodermal influence on physiological cell death in the posterior necrotic zone of the chick wing bud.

The phenomenon of "programmed cell death" in the posterior necrotic zone (PNZ) of the chick wing bud was reexamined. Prospective PNZs (pPNZs) were excised from stage 18-21 donor wings and observed for signs of necrosis in vitro. Cell death was quantified by a chromium-51 release assay. Prospective PNZs from the youngest donors (stage 18) showed no signs of death above control levels, while necrosis increased in vitro with increasing donor age. Cell death in the PNZ at stage 24 could be inhibited by removing the overlying ridge at stage 20 or 21. These results suggest that cell death in the PNZ is not rigidly determined early in development as previous studies suggest, but remains responsive to the cellular environment until shortly before the cells die.

The ectodermal control of mesodermal patterns of differentiation in the developing chick wing.

The influence of limb ectoderm on the dorso-ventral muscle and skeletal patterns in the chick wing was studied by recombining stage 14-21 limb mesoderm with the same stage ectoderm in dorso-ventrally reversed orientation. Recombinants grafted to the flank of host embryos were allowed to develop for 10 days. Fully developed wings obtained from stage 15-21 donor embryos have at their distal half d-v polarity conforming to the reversed ectoderm and proximally polarity conforming with the mesoderm. The ectodermal effect is generally observed as a bidorsal feather pattern at the autopod and an almost complete d-v reversal of muscle and skeletal patterns. In experimental wings from donor embryos younger than stage 15, the dorso-ventral pattern conforms with the polarity of the limb mesoderm. The results suggest that control of dorso-ventral polarity resides in the mesoderm until the onset of limb development at stage 15. At this stage, the ectoderm acquires dorso-ventral information which it can impose on the mesoderm.

Pattern regulation along the proximodistal axis of the developing chick limb.

Transplants creating presumptively excess or deficient proximodistal segments were performed to investigate pattern regulation in stage 21 and stage 23 chick limb buds. Excess skeletal elements were not removed by regulation. Deficiencies appeared to be regulated by host tissue. The ability of the host tissue to participate in regulation declined in more proximal tissue and with increased age. These results suggest that there are two requirements for pattern regulation. First, the tissue in question must be sufficiently close to the ectodermal ridge before it can respond. Results of previous work and implications for pattern formation are discussed with these requirements in mind.

Verification of an in vitro bioassay for limb bud polarizing activity.

An in vitro bioassay for limb bud polarizing activity in the chick embryo has been verified by two procedures, demonstrating that the culture procedure mimics occurrences in vivo. First, no activity can be detected with the in vitro assay 24 hours after removal of the posterior region of the limb. In addition, after a positive assay for activity, the responding tissue develops into polarized limb structures when transplanted to a host embryo. After a negative assay, the transplanted responding tissue fails to develop into recognizable limb structures. Since polarizing activity is defined by its ability to induce polarized limb structures in vivo we conclude that the in vitro system provides a valid assay for limb polarizing activity.

The target tissue of limb-bud polarizing activity in the induction of supernumerary structures.

When polarizing mesoderm from the posterior border of the 4-day chick limb bud is placed adjacent to anterior limb mesoderm and ectodermal ridge, the anterior ridge thickens and mesodermal outgrowth ensues, resulting in supernumerary limb structures. This apposition of anterior and posterior limb tissues can be accomplished by cutting off the apical one third of the limb bud and reimplanting it on the stump with its anteroposterior axis reversed. The preaxial response to polarizing activity can be obtained after only 12--18 h in the reoriented position. Reversed apical mesoderm develops supernumerary digits when combined with untreated ectoderm. The reciprocal combination, reversed ectoderm and untreated mesoderm, fails to develop supernumerary structures. We have interpreted this as evidence that, in inducing supernumerary limb structures, polarizing activity acts only on the mesoderm.

Polarizing activity in the developing limb of the Syrian hamster.

The transplantation of small pieces of tissue from the limb buds of 9 1/2 -10 day hamster embryos to the wing bud of the chick results in the induction of supernumerary wing structures. The anteroposterior polarity of these induced structures is under the control of the transplanted hamster tissue. The developing hamster limb thus has limb polarizing activity similar to that found in avian species and, as in the chick, the activity is found primarily in the posterior region of the limb bud.

Limb development in diplopodia4: a polydactylous mutation in the chicken.

In a study of the polydactylous mutation of the domestic chicken, diplopodia4, we have found that the genetic lesion affects primarily the mesoderm and only secondarily the ectoderm. The effect of this mutant mesenchyme on overlying ectodermal ridge, either mutant or normal, is to thicken the ridge preaxially, leading to increased outgrowth and preaxial polydactylism. A "zone of polarizing activity" in the normal limb-bud seems to have a role in the control of its anteroposterior polarity. We have examined diplopodia4 limb-buds for polarizing activity and found it to be normal in its activity and distribution. These results suggest that the supernumerary outgrowth in the mutant limbs result from increased ridge mainon of the polarizing zone.