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.     

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

Summary 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 (FGF-2) rescue in the OP and PNZ 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.     

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.     

Onset of troponin synthesis in the chick wing bud.

Indirect antibody labeling techniques were used to determine when cells in the chick embryo wing bud begin to synthesize troponin. Frozen sections of stage 22 through stage 27 wing buds were treated with antibodies to the troponin complex and fluorescein-labeled antiimmunoglobulin. Cells producing detectable quantities of troponin were found first in late stage 24 or early stage 25 wing buds; all wing buds stage 25 and older contained labeled cells. Cells synthesizing troponin were initially localized in the muscle-forming areas of the wing bud nearest to the body wall. As the wing bud developed, cells located in more distal areas of the wing bud became labeled with fluorescent antibody, and the number of cells engaged in troponin synthesis increased in all areas. At all stages in which labeling occurred, some cells contained fluorescent cross-striations. When placed in the context of recent studies on the appearance of myofibrillar proteins, these results indicate that myogenic cells in the chick limb bud begin to synthesize large quantities of troponin at approximately the same time as the other muscle contractile proteins.     

Supernumerary limb structures after juxtaposing dorsal and ventral chick wing bud cells

The formation of duplicated wing skeletal elements and/or extra wing muscles was studied by juxtaposing normally nonadjacent embryonic chick wing bud cells. A wedge of right or left stage 21 wing bud ectoderm and mesoderm was inserted in a slit made in a host stage 20 to 22 right wing bud at the same anteroposterior position as its position of origin. The distal edge of the donor wedge and host wing bud were aligned with each other. Donor tissue was grafted into a host wing bud in one of the following four axial relationships: both the anteroposterior and dorsoventral axes corresponded with each other (aadd); only the anteroposterior axes were opposed (apdd); only the dorsoventral axes were opposed (aadv); both the anteroposterior and dorsoventral axes were opposed (apdv). Of the 63 wings resulting from the control aadd operation and the 45 wings from the apdd operation, only 12 wings had a duplicated skeletal element; of the 69 wings sectioned from these two groups of operations, only one had an extra muscle. However, of the wings resulting from the aadv and apdv operations (48 and 52 cases, respectively), 23 had a duplicated skeletal element; of the 54 wings sectioned from these operations, 43 wings had one to four extra muscles. Furthermore, when the aadv operation was performed with a wedge of donor quail wing bud ectoderm and mesoderm or mesoderm alone, supernumerary muscles formed in these chimeric wings and they were made up of donor quail and host chick cells or only donor quail cells.     

The formation of leg or wing specific structures by leg bud cells grafted to the wing bud is influenced by proximity to the apical ridge

When quail or chick leg bud mesoderm was grafted to a chick wing bud, toes developed from grafts placed in direct contact with the wing apical ridge. The toes were primarily derived from quail leg cells, with variable participation of host wing cells. Donor cells also integrated into wing-specific structures, such as cartilage of the wing digits and the surrounding connective tissues. In addition to forming toes, the grafted leg mesoderm expressed its leg origin by enlarging skeletal elements in the host wing. In all cases, enlargements were derived of both quail donor and chick host cells, and were not the result of the addition of mass to the host bud. Grafts placed further than 162 microns from the ridge formed neither toes nor enlargements; rather, they integrated into wing-specific structures. Under the influence of the apical ridge, the grafted leg mesoderm cells are able to maintain their leg character and to form toes and skeletal enlargements. Grafts outside the range of ridge influence (162 microns) are affected by their surroundings to integrate into wing-specific structures. The formation of leg-specific structures by leg bud mesoderm grafted to the wing bud has been used to support the principle of nonequivalence, which states that, because of their different developmental histories, wing and leg cells are restricted to form structures specific for their respective limbs. However, we have shown that leg cells can form wing-specific structures, and therefore limb cells are not restricted in their development.     

Hensen's node,but not other biological signallers,can induce supernumerary digits in the developing chick limb bud

Summary The purpose of this study was to determine whether the organizer regions of early avian and amphibian embryos could induce supernumerary (SN) wing structures to develop when they were grafted to a slit in the anterior side of stage 19–23 chick wing buds. Supernumerary digits developed in 43% of the wings that received anterior grafts of Hensen's node from stage 4–6 quail or chick embryos; in addition, 16% of the wings had rods of SN cartilage, but not recognizable SN digits. The grafted quail tissue did not contribute to the SN structures. When tissue anterior or lateral to Hensen's node or lateral pieces of the area pellucida caudal to Hensen's node were grafted to anterior slits, the wings usually developed normally. No SN structures developed when Hensen's nodes were grafted to posterior slits in chick wing buds. Wings developed normally when pieces of the dorsal lip of the blastopore from stage 10–11.5 frog (Xenopus laevis and Rana pipiens) embryos were grafted to anterior slits. No SN digits developed when other tissues that have limb-inducing activity in adult urodele amphibians [chick otic vesicle, frog (Rana pipiens) lung and kidney] or that can act as heteroinductors in neural induction (rat kidney, lung, submaxillary gland and urinary bladder; mouse liver and submaxillary gland) were grafted to anterior slits in chick wing buds. SN digits also failed to develop following preaxial grafts of chick optic vesicles. These results suggest that although the anteroposterior polarity of the chick wing bud can be influenced by factors other than the ZPA (e.g., Hensen's node, retinoids), the wing is not so labile that it can respond to a wide variety of inductively-active tissues.     

Analysis of type II collagen RNA localization in chick wing buds by in situ hybridization

Type II collagen is a major component of cartilage extracellular matrix. Differentiation of mesenchyme into cartilage involves the cessation of type I collagen synthesis and the onset of type II collagen synthesis. Solution hybridization of mRNA isolated from chick limb buds with a cDNA probe to type II collagen mRNA showed the presence of small amounts of type II collagen message in mesenchymal chick limbs. We have examined the localization of type II collagen mRNA in mesenchymal chick wing buds by in situ hybridization using single stranded RNA probes. Our results show a small but detectable amount of type II collagen RNA distributed uniformly in early limbs until the first precartilage condensations form at stage 22. This is interesting because it is known that mesenchyme isolated from chick wing buds has the capacity to undergo chondrogenesis in culture, even if taken from nonchondrogenic areas of the limb. At stage 23, type II collagen mRNA is found at significantly increased levels in the cells of the precartilage condensation when compared to the other limb cells. As chondrogenesis proceeds, the amount of type II collagen RNA increases even more in cells of the cartilage elements. The signal in the peripheral tissue is indistinguishable from background. These results show that type II collagen message exists at low levels in cells throughout the mesenchymal chick wing bud, until the formation of the condensation results in an elevation of type II mRNA in the prechondrogenic cells found in the core of the limb.     

Temporal pattern of posterior positional identity in mouse limb buds

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.     

The preservation of the ability of cultured quail wing bud mesoderm to elicit a position-related differentiative response

A previous study showed that grafting wedges of fresh anterior quail wing mesoderm into posterior slits of chick wing buds resulted in the formation of rods and nodules of cartilage in a high percentage of cases (B. Carlson, 1983, Dev. Biol. 101, 97-105). The purpose of the present study was to determine if a similar response could be elicited by grafting pieces of mesoderm that had been cultured in vitro. When pieces of 1-day cultured anterior mesoderm from stage 17-24 donors were grafted into standard posterior slits of chick wing buds, the percentages of supernumerary structures differed little from those which formed after the grafting of pieces of fresh mesoderm. In a time series, grafts of stage 22-23 anterior mesoderm which had been cultured for 1-4 days retained the ability to form cartilage after being grafted into posterior locations. A time series showed that the duration of this retention was longer in cultured mesoderm than it was in mesoderm that remains in the donor wing bud.     

Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.     

Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms.

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5'-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.     

Régulation des excédents dans le développement du bourgeon de membre de l'embryon d'oiseau. Analyse expérimentale de combinaisons xénoplastiques caille/poulet

Summary In order to support the demonstration of the regulative capacity of the chick limb bud, already stressed by one of us (Kieny, 1964, 1967), heterospecific combinations were made between chick and quail tissues, the cells of the latter bearing a distinctive nuclear marker. A Japanese quail whole limb bud (stage-18 to 21 of H. H., wing or leg) was grafted distally onto the prospective zeugopod of a chick (stage-22) wing bud sectioned at the prospective wrist level. Thus, from a heterospecific surplus recombinant containing five prospective limb segments (stylopod and zeugopod from the chick host; stylopod, zeugopod and autopod from the quail graft), it was possible to obtain a normally shaped appendage that comprised either upper arm, lower arm and hand in the case of a wing bud graft, or heteromorphic upper arm, lower leg and foot in the case of a hind-limb bud graft. In these cases, regulation for excess appeared to take place mainly within the host tissues. The three proximal segments of the recombinant, namely the chick stylopod and zeugopod of the host's stump and the quail stylopod of the graft, became reorganized and gave rise to a single stylopodial segment, which usually contained a double stylopodial bone element, one of chick, the other of quail origin.The absence of development of the squeezed prospective zeugopod can be interpreted as follows: owing to an interaction with the stylopodial graft tissues, the zeugopodial cells of the juxtaposed stump boundary have shifted proximally their originally more distal positional values, so that they changed their prospective pattern of differentiation to that of stylopod. These reset zeugopodial cells combine with the stylopodial cells of host and graft and form a huge composite stylopod, in which, due to an asynchronous determination in the two species, chick and quail tissues do not cooperate fully for the development of a single bone.

Ce travail a été effectué avec l'aide de la D.G.R.S.T. (Action complémentaire coordonnée: Biologie de la reproduction et du développement, convention n^o 73-7-1661)     

Preservation of the ability of dissociated quail wing bud mesoderm to elicit a position-related differentiative response

Previous studies showed that grafting wedges of fresh or cultured anterior quail wing mesoderm into posterior slits in chick wing buds resulted in the formation of supernumerary cartilage in a high percentage of cases. When anterior quail mesoderm, which had been dissociated into single cells and pelleted by centrifugation, was grafted into posterior slits of host chick wing buds, supernumerary rods or nodules of cartilage formed in 74.3% of the cases. Few supernumerary skeletal structures formed following control operations in which pelleted dissociated anterior or posterior mesoderm was grafted into homologous locations in host chick wing buds. When pelleted, dissociated anterior mesoderm was cultured in vitro for 1 or 2 days prior to being implanted in posterior locations, the incidence of supernumerary cartilage formation increased to 95.5% and 93.8%, respectively. The incidence of supernumerary cartilage formation following control orthotopic grafts of cultured mesoderm was 11.8% for 1-day and 31% for 2-day cultured anterior mesoderm; for 1- and 2-day cultured posterior mesoderm, the incidence of supernumerary cartilage formation was 20% and 41.7%, respectively. Longer-term culture resulted in a substantial decrease in the percentage of supernumerary cartilage after anterior to posterior grafts and an increase in the incidence of supernumerary cartilage from control grafts. The results demonstrate that quail anterior wing bud mesodermal cells do not need to maintain constant contact with one another in order to retain the ability to form or stimulate the formation of supernumerary cartilage after being grafted into a posterior location in a host wing bud. This ability is retained when the pelleted dissociated mesoderm is cultured in vitro outside the limb field for at least 1 to 2 days.     

Retinoic acid respecifies limb bud cells in vitro.

Retinoic acid (RA) is known to have dramatic effects on limb pattern formation and has been shown to exert its effects on limbs by converting anterior limb bud cells into cells with posterior positional properties. In this study we find that dissociated posterior limb bud cells from chick and mouse embryos cultured at high density (micromass cultures) are able to stimulate the formation of supernumerary digits when grafted into developing wing buds and that the positional identity of both chick and mouse limb bud cells can be maintained for finite periods of time in vitro. Furthermore, using this assay system we have tested whether anterior cells from mouse and chick limb buds can be converted into cells with posterior identity by exposure to RA in vitro. We find that anterior limb bud cells acquire posterior properties after culture in the presence of RA.     

Anti-Bcl-2 family members,zfBcl-x<Subscript>L</Subscript> and zfMcl-1a,prevent cytochrome <Emphasis Type="Italic">c</Emphasis> release from cells undergoing betanodavirus-induced secondary necrotic cell death

Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 μg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 μg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein α, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x_L and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members. J.-L. Wu and J.-R. Hong contributed equally to the research.     

The role of the zone of polarizing activity in controlling the differentiation of the apical mesenchyme of the chick wing-bud: histochemical thechniques in the analysis of a developmental problem

Summary Following excision of the posterior half of the three-day chick wing bud, the anterior half which normally forms humerus (part), radius and digit 2, forms only a single skeletal element (humerus, of humerus fused with reduced radius). If part of the zone of polarizing activity is included in the remaining anterior part of the wing bud, a normal wing with normal skeleton forms. Excision of the anterior half of the chick wing-bud results in the posterior half forming humerus (part), ulna and digits 3–5. This is confirmed as the normal prospective fate of the posterior half by chimeric quail-chick wing-buds in which Feulgen staining of the nucleolus-associated heterochromatin of quail cells enables their contribution to the resultant skeleton to be identified. Beginning at 18 h alter posterior half amputation, the anterior distal mesenchyme becomes necrotic and the apical e todernal ridge regresses. By contrast, following anterior half amputation, posterior halves develop no more cell death than control wing-buds.Anterior half regression is characterized by cell fragmentation and phagocytosis. First, in both the apical ectodermal ridge and distal mesenchyme cells, acid phosphatase-rich autophagic bodies appear, the cells then becoming autolytic (with diffuse acid phosphatase activity) and fragmenting. Neighbouring cells phagocytose the dead cell fragments, the mesenchyme cells forming large non-professional macrophages containing many acid phosphatase-rich vacuoles.These experiments show that for survival and differentiation, the anterior and distal mesenchyme of the wing bud requires a factor from the posterior part, thus suggesting that the zone of polarizing activity controlsantero-posterior differentiation in the normal wing.     

Experimental manipulation leading to induction of dorsal ectodermal ridges on normal limb buds results in a phenocopy of the eudiplopodia chick mutant

Elongation of chick limb buds depends on the presence of the apical ectodermal ridge which is induced by subjacent limb bud mesoderm. Recombination experiments have shown that the limb bud mesoderm loses the capacity to induce ridges by late stage 17. Moreover, in normal limb development only one ridge forms. However, in the eudiplopodia chick mutant accessory ectodermal ridges form on the dorsal surface of limb buds as late as stage 22. Tissue recombinant experiments show that the mutation affects the ectoderm, extending the time it responds to ridge induction (Fraser and Abbott, 1971a, Fraser and Abbott, 1971b while the mesoderm is normal. The result is polydactyly, with extra digits dorsal to the normal digits. Because eudiplopodia limb bud dorsal mesoderm can induce ridges at stage 22 but is unaffected by the gene, genetically normal dorsal limb bud mesoderm may also be able to induce ridges after stage 17. To test this possibility we grafted stages 14–18 flank ectoderm to normal limb bud dorsal mesoderm and found that mesoderm from stages 17 through 20 was able to induce a ridge and subsequently dorsal digits developed. Limbs with duplicate digits were similar to eudiplopodia limbs. In other experiments, stage 18, 19, and 20 leg bud dorsal ectoderm did not form ridges when grafted to leg bud dorsal mesoderm of the same stage, indicating a lack of response to the mesoderm. Finally, the inductive capacity of limb bud mesoderm appeared to be reduced compared to mesoderm at pre-limb bud stages. These experiments demonstrate a spatially generalized potential in limb bud dorsal mesoderm to induce ridges during the stages when the apical ridge is induced. The determination of where the ridge will form and the acquired inability of limb bud dorsal ectoderm to respond to induction by underlying mesoderm are necessary early pattern forming events which assure that a single proximodistal limb axis will form.