A test of positional properties of avian wing-bud mesoderm

Supernumerary wing structures are readily produced by grafting pieces of wing-bud mesoderm into different locations of host wing buds, but the mechanism underlying their formation remains obscure. The major aim of this study was to examine the ability of posterior quail wing-bud mesoderm, cultured in vitro long enough to lose ZPA (zone of polarizing activity) activity, to stimulate or participate in the formation of supernumerary structures when grafted into anterior slits of host chick wing buds. Small pieces of anterior and posterior quail wing-bud mesoderm (HH stages 21-23) were placed in in vitro culture for up to 3 days. After 2 days, ZPA activity of cultured mesoderm was lost. After the grafting of 2- to 3-day cultured anterior quail wing-bud mesoderm into posterior slits of host chick wing-buds, a consistently high percentage (70%-90%) of grafts result in formation of supernumerary cartilage; in this experiment, however, only a low percentage of grafts resulted in supernumerary cartilage when 2- to 3-day cultured posterior mesoderm was grafted into anterior slits. Taken with controls, these results show that positional differences exist between cultured anterior and posterior wing-bud mesoderm. Serial-section analysis of numerous operated wings has shown several patterns of contribution to supernumerary structures by cells of graft and host. Single supernumerary digits induced by grafts of ZPA mesoderm into anterior slits were normally composed entirely of host cells, but graft cells regularly contributed to skeletal elements of more complex supernumerary structures. Cartilage rods produced by anterior-to-posterior grafts were composed mostly of graft cells, but cartilage nodules and the bases of some rods were often mosaics of chick and quail cells. The results support the proposition that mesodermal cells of the quail wing-bud possess a form of anteroposterior positional memory, but its nature and the means by which the memory of grafted cells interacts with host mesoderm are still not clear.     

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.     

The formation of supernumerary structures after grafting anterior quail wing bud mesoderm of various ages into chick wing buds

Wedges of anterior quail mesoderm grafted into posterior slits in the wing buds of chick embryo hosts result in the formation of rods and nodules of supernumerary cartilage in a high percentage of cases. Identifiable digits do not form unless the ectoderm is allowed to remain on the grafts. Control experiments have shown that wedges of anterior or posterior wing mesoderm placed into homologous locations of host wing buds produce few or no supernumerary skeletal structures. Anterior-to-posterior grafts of stage 17 mesoderm evoke a 71.4% incidence of supernumerary cartilage. This percentage increases to 100% with stage 22 donor mesoderm. The percentage of supernumerary structures formed declines markedly with donor mesoderm of stages 24-30. By stages 35-36, only 10% of the grafts result in the formation of supernumerary structures. The period of decline coincides with the onset of overt cytodifferentiation within the donor mesoderm.     

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.     

A comparative study of myogenic cell invasion of the avian wing and leg bud.

The borders of myogenic cell invasion of avian wing and leg buds were determined using the interspecific grafting technique between quail and chick embryos. Distal parts of quail limb buds were grafted ectopically into the coelomic cavity of chick embryos. The presence or absence of skeletal muscle was investigated in histological sections of the reincubated grafts. A comparison between the borders of myogenic cell invasion of the wing and leg buds showed that the differences in the position of the distal most muscles in the adult avian limbs could be a consequence of the cranio-caudal sequence of development.     

The determination of myogenic and cartilage cells in the early chick embryo and the modifying effect of retinoic acid

Summary The time of determination of cartilage and skeletal muscle was studied by making chimeric grafts or explants of small tissue pieces from several stages of early chick or quail embryos. Chondrogenesis was assessed by histology or with antibodies directed against type II collagen or cartilage proteoglycan, while myogenesis was detected immunohistochemically with antibodies directed against 3 different muscle markers, including muscle myosin. Grafts from Hensen's node, primitive streak and segmental plate of donor embryos of Stage 3–5 (Hamburger and Hamilton) were transplanted under the ectoderm in the extraembryonic area of Stage 12 host embryos. In addition, explants and mesodermal cells were cultured on glass in DMEM+F12 medium supplemented with 10% FCS. The results showed that determined myogenic cells could first be detected in Hensen's node and the primitive streak at Stage 3⁺–4 and that they developed from mesodermal cells located between the epiblast and hypoblast. Myogenic cells also appeared in grafted and explanted segmental plate with or without notochord from Stage 5 embryos. On the other hand, cartilage cells only formed in grafted and explanted segmental plate that also contained notochord. RA (1 ng/ml) could induce the formation of cartilage cells in the explanted primitive streak without Hensen's node or notochord taken from Stage 3–5 embryos and could also promote the differentiation of myogenic cells in primitive streak from Stage 3 embryo. Thus RA can substitute for Hensen's node or the notochord in the induction of cartilage cells and has some stimulatory effects on the differentiation of myogenic cells. Additional evidence indicates that the hypoblast might play an inductive role in the formation of the notochord which may subsequently promote the differentiation of cartilage cells. Offprint requests to: M. Solursh     

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.     

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.     

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.     

Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds.

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is "posteriorized" and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pigment patterns in neural crest chimeras constructed from quail and guinea fowl embryos

The pattern of pigmentation in bird embryos is determined by the spatial organization of melanocyte differentiation. Some of the results from recent, neural crest transplantation experiments support a model based on a prepattern in the feathers; others could be interpreted in terms of a nonspecific pattern resulting from a failure of the crest cells to read the positional values in another species. To distinguish between these possibilities, the crucial test is to construct chimeras from two species with different pigment patterns. We have examined the wing plumage of quail and guinea fowl embryos. The quail has a characteristic pattern of pigmented and unpigmented feather papillae, whereas the guinea fowl shows uniform pigmentation. Chimeras were constructed by grafting wing buds isotopically between embryos. The wing buds were transplanted before they had become invaded by neural crest cells. Quail wing buds grafted to the guinea fowl developed, in most cases, a pigment pattern resembling that of the quail and not that of the guinea fowl. A few cases became uniformly pigmented and appeared to represent nonspecific patterns. The reciprocal grafts (guinea fowl wing buds grafted to the quail) became pigmented all over. We found evidence that the timing of melanocyte differentiation is controlled by cues in the feather papillae. Some cases developed a severe inflammatory response. The model which best accounts for these findings--and which can account for inconsistencies in previous reports--is the following. A prepattern is present in the feathers and this can control the differentiation of melanoblasts, even if they come from a different species. The local cues which constitute the prepattern are not positional values. In some chimeras melanoblasts fail to respond to the prepattern and so a nonspecific pattern of uniform pigmentation is produced.     

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.     

Retinoic acid induces a pattern of digits in anterior half wing buds that lack the zone of polarizing activity

Wing buds whose posterior half is excised, develop into wings lacking distal structures. However, such experimentally generated preaxial half wing buds can be rescued by implanting a retinoic-acid-releasing bead at their anterior margin. The polarity of the pattern that originates from preaxial half wing buds is reversed. For example, instead of a 234 digit pattern typical for normal wings, the order of digits is 432. This result implies that retinoic acid has the capacity to reprogram anterior limb bud tissue, and that the resulting change in cell fate does not depend on the presence of posterior tissue regions such as the zone of polarizing activity (ZPA).     

A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

The spatial and temporal distribution of polarizing activity in the flank of the pre-limb-bud stages in the chick embryo

The presence of polarizing activity in the limb buds of developing avian embryos determines the pattern of the anteroposterior axis of the limbs in the adult. Maps of the spatial distribution and the strength of the signal within limb buds of different stages are well documented. Polarizing activity can also be found in Hensen's node in the early embryo. We have mapped the distribution of polarizing activity as it emerges from Hensen's node and spreads into the flank tissue of the embryo. There is a clear change in the local pattern of expression of polarizing activity between stage 8 and 18. Almost no activity is measured for stages 8 and 9. More or less uniform levels of around 10% are spread along the flank lateral to the unsegmented somitic mesoderm from somite position 12 to 22 in stage 10 embryos. Some 6 to 8 h later at stage 12, there is a distinct peak of activity at somite position 18, the middle of the wing field. This peak increases at stages 13 to 15 and its position traverses to the posterior edge of the wing field. Full strength of activity is reached shortly before the onset of limb bud formation at stage 16 to 17. Stages 16 to 18 were investigated for polarizing activity in the wing and the leg field. Low levels of polarizing activity are present in the anterior leg field at stages 16 and 17 but have disappeared by stage 18 and all activity is confined to the posterior part of the leg bud.     

A study of the development of the hemopoietic system using quail-chick chimeras obtained by blastoderm recombination

The anterior part of the area pellucida from quail blastoderms extending to the 10th or the 17th somite level was substituted for the corresponding region of chick blastoderms in ovo. Reciprocal grafts were also carried out. In external appearance the resulting chimeras had a composite body, one species contributing the head and neck or the head, neck, and wing regions while the other species provided the remainder. The chimeras were always grafted on a chick yolk sac. The cellular composition of hemopoietic organs according to species was analyzed by means of the quail-chick nuclear difference, in 39 viable chimeras at 11–13 days of incubation. The thymus composition depended on the level of the boundary between the two species. In chimeras in which the quail contributed head and neck, the thymic epithelial stroma was made of quail cells while the lymphoid population was of chick origin. In contrast, when the quail contribution also extended to the wings, both thymic stroma and lymphoid cells were of quail origin. In reciprocal combinations, in which head and neck were of chicken origin on a quail body, a different result was obtained: no lymphoid cells were present in the thymus which was reduced to its epithelial component and this was of chick origin. On the other hand, if the chick contribution extended to the wings, as in the reciprocal combination, all thymus components were of chick origin. The spleen and the bursa of Fabricius in most instances did not differ in their cellular composition from the surrounding tissues; however in some chimeras a minor admixture of cells of the other species was also found. Overall these results suggest that hemopoietic stem cells destined to colonize intraembryonic organs arise in territories derived from the whole area pellucida excluding the prospective head-neck region. Furthermore, each hemopoietic organ rudiment appears to be colonized by precursors derived from adjacent territories.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells

Using chimeras consisting of chick embryos that had received substitution grafts of quail somites, we have determined the distalmost extension of the myogenic primordia in the outgrowing wing bud at 5 days of incubation. At Hamburger-Hamilton stage 25 the most distal premuscle cell is consistently 300 mum or more from the apex of the wing mesoblast. The stage 25 wing tip resembles very early whole limb buds in not having proceeded beyond the mesenchymal state or having expressed markers of terminal differentiation. However, unlike early whole limb buds it is free of a myogenic subpopulation. We therefore propose that the stage 25 wing tip is the appropriate system for in vitro and molecular studies of cartilage differentiation.     

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.