Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re-expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.     

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.     

Limbness in the early chick embryo lateral plate

In order for the limb to be useful in the evaluation of early determinants of morphogenesis, it is necessary to understand some of the characteristics associated with "limbness" and, more importantly at the beginning at least, it is necessary to know what regions of the early embryo exhibit limbness qualities. Previous investigators have assumed, without direct experimental evidence, that the flank does not have limbness qualities, even at early stages of development. However, there are a few studies suggesting that the early flank does possess limbness qualities. The purpose of the present study was to determine how extensively the qualities of limbness exist in the early chick embryo. Tissues from the future neck, wing, flank, and leg regions were grafted to host celoms and evaluated for their abilities to form limbs. Limbs developed from all four regions of stage 11-14 embryos, but after stage 14 only grafts from the wing and leg regions formed limbs.     

Normal development of the chick wing following removal of the polarizing zone.

The negative results of assays for polarizing activity along the posterior border of the chick wing 24 and 48 hours after removal of the polarizing zone demonstrate that this zone is not regenerated following removal. These results, and the fact that normal wing development can occur after polarizing zone removal from stages 15 through 24 wing buds, indicate that during these stages the polarizing zone has no direct role in normal development of the limb bud. It is speculated that the polarizing zone is effective only during limb induction and that after this time it exists in latent or residual form.     

A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development

Small, positively charged beads that slowly release known amounts of all-trans-retinoic acid have been implanted below the apical ectodermal ridge at the anterior margin (opposite somite 16) of wing buds of 3 1/2 day-old chick embryos. The continuous release of retinoic acid is shown to create an anteroposterior concentration gradient of retinoic acid in the limb field that is stable with time, despite the fact that this compound is metabolized by the limb tissue. With beads that release increasing amounts of retinoic acid, the normal 234 digit pattern is progressively altered to a 2234, to a 32234, and then to a 432234 pattern. The tissue concentrations of all-trans-retinoic acid required to change the digit pattern in this way range between 1 and 25 nM. When the same amounts of retinoic acid are released from posteriorly implanted beads (placed below the apical ectodermal ridge opposite somite border 19/20 or somite 20), the normal digit pattern is unaffected. Implantations of beads that release all-trans-retinoic acid are thus identical in their effect to grafts of cells from the limb polarizing region, which cause similar dose-dependent changes in the digit pattern when grafted to the anterior margin of the bud (but not when grafted opposite somites 19 or 20). Because of the low concentrations of retinoic acid required for its biological effect, the graded response observed, and the fact that a concentration gradient is established across the limb field, all-trans-retinoic acid closely mimics the putative morphogen that has been postulated to be emitted by polarizing region cells during normal development.     

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.     

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.     

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.     

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.     

Inhibitory effect on limb morphogenesis by cells of the polarizing zone coaggregated with pre- or postaxial wing bud mesoderm.

The influence of cells of the polarizing zone mesoderm on the morphogenesis of recombinant chick limbs was studied. The recombinant buds were composed of leg bud ectoderm and different regions of the wing bud mesoderm, which had been dissociated and reaggregated. In any case where the polarizing zone mesoderm was coaggregated with the wing mesoderm the morphogenetic capabilities of the recombinant were reduced. This was the case with postaxial mesoderm, preaxial mesoderm plus polarizing tissue, and postaxial mesoderm from which a piece of the nonpolarizing mesoderm (comparable in size to the polarizing zone) had been removed. All of these gave outgrowths with digits in only a very low percentage of cases. In contrast, those recombinants without polarizing mesoderm developed outgrowths with digits in a high percentage of cases, indicating good morphogenesis. Finally, if the polarizing zone were removed prior to dissociation, the recombinant limb, composed of the total remaining wing bud mesoderm plus leg bud ectoderm, exhibited a higher percentage of complete morphogenesis than if the polarizing zone had been part of the recombinant.It is clear that cells of the polarizing zone, when dissociated, and coaggregated with wing mesoderm, are inhibitory to the morphogenetic performance of that mesoderm in the recombinant limb situation.     

The in vitro maintenance of the apical ectodermal ridge of the chick embryo wing bud: an assay for polarizing activity.

We have devised an in vitro bioassay for limb bud polarizing activity in the chick embryo. This assay has proven to be a relatively quick and effective test for a morphogenetic factor asymmetrically distributed in the limb bud which is capable of maintaining or thickening the apical ectodermal ridge.A small section of the preaxial border of the chick embryo wing bud was cultured alone, with tissue from the posterior border, mid-dorsal or anterior corner of a second donor wing, or from the flank. The tissue from the preaxial border (responding tissue) consisted of mesoderm with overlying ectoderm and apical ectodermal ridge. When the responding tissue was cultured alone, with flank, or with anterior corner limb tissue, the apical ectodermal ridge flattened in 24–36 hr and many macrophages appeared in the underlying mesoderm. When cultured with posterior border limb tissue however, the apical ridge of the responding tissue remained thickened for up to 48 hr., and no macrophages appear in the underlying mesoderm. The behavior of responding tissue was intermediate between these two extremes when cultured with mid-dorsal limb tissue. The morphogenetic activity assayed by this procedure thus seems to be present as a gradient in the wing bud, with activity decreasing from posterior to anterior. Contact with the responding tissue is not required to enable posterior border tissue to elicit ridge thickening and inhibit the cell death.     

Altered expression of the chicken homeobox-containing genes GHox-7 and GHox-8 in the limb buds of limbless mutant chick embryos.

It has been suggested that the reciprocal expression of the chicken homeobox-containing genes GHox-8 and GHox-7 by the apical ectodermal ridge and subjacent limb mesoderm might be involved in regulating the proximodistal outgrowth of the developing chick limb bud. In the present study the expression of GHox-7 and GHox-8 has been examined by in situ and dot blot hybridization in the developing limb buds of limbless mutant chick embryos. The limb buds of homozygous mutant limbless embryos form at the proper time in development (stage 17/18), but never develop an apical ectodermal ridge, fail to undergo normal elongation, and eventually degenerate. At stage 18, which is shortly following the formation of the limb bud, the expression of GHox-7 is considerably reduced (about 3-fold lower) in the mesoderm of limbless mutant limb buds compared to normal limb bud mesoderm. By stages 20 and 21, as the limb buds of limbless embryos cease outgrowth, GHox-7 expression in limbless mesoderm declines to very low levels, whereas GHox-7 expression increases in the mesoderm of normal limb buds which are undergoing outgrowth. In contrast to GHox-7, expression of GHox-8 in limbless mesoderm at stage 18 is quantitatively similar to its expression in normal limb bud mesoderm, and in limbless and normal mesoderm GHox-8 expression is highly localized in the anterior mesoderm of the limb bud. In normal limb buds, GHox-8 is also expressed in high amounts by the apical ectodermal ridge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vital labelling of somite-derived myogenic cells in the chicken limb bud

Summary In order to understand how myogenic cells migrate in the limb bud, it is indispensable to distinguish undifferentiated myogenic cells from other mesenchymal cells. Thus, a suitable method for this purpose has been sought. A method to exchange the somites of a chicken and a quail microsurgically has widely been used, since the nuclei of the two species are morphologically distinguishable. However, microsurgery is accompanied by disturbances at the operated locus, and introducing cells of different species might induce unexpected effects. We report a new method for labelling chicken myogenic cells without transplantational operations, and describe their migration pattern in limb buds. Injection of a fluorescent carbocyanine dye into the somite lumen intensely labelled the somitic cells. Myogenic cells derived from the somite were clearly detected in limb buds. Before stage 20, the labelled cells were diffusely distributed in the proximal region of the limb bud. At about stage 21 in both wing and leg buds, labelled cells began to form dorsal and ventral masses. The label was followed until the cells differentiated and expressed myosin. This vital labelling method has advantages over the somite transplantation method: it does not include surgical operations that may disturb the normal development, and the cells are labelled intensely enough to be detected in a whole mount preparation. Offprint requests to: K. Hayashi     

Temporal and spatial expression of TGF-beta2 in chicken somites during early embryonic development

A multifunctional growth and differentiation factor TGF-beta is expressed at various developmental stages, and its principle role may be involvement in organogenesis. The present study was performed to evaluate the temporal and spatial expression of TGF-beta2 mRNA in developing somites of chicken embryos during their early developmental periods. TGF-betas were expressed in various tissues of the whole embryo obtained at stage 26 (5 days of incubation) as revealed by whole-mount in situ hybridization. TGF-beta2 mRNA was predominantly expressed in somites as well as the head, branchial arch, wing buds, and leg buds. TGF-beta2 mRNA first appeared in the rostral somites on E4, and its expression sites expanded to the middle range of somites at stage 26. At stages 29-31 (6-7 days), expression in the rostral somites disappeared, and it appeared in the caudal somites. TGF-beta2 expression was also analyzed in sections of the embryo by in situ hybridization. The expression sites of TGF-beta2 were clearly observed in the myotomal somite tips as well as the neural tube. RT-PCR analysis showed that TGF-beta2 expression was very low in the blastocyte stage embryo and thereafter increased linearly in the whole trunk until stage 26. These data indicate that TGF-beta2 may be a regulatory factor participating in the somitogenesis of chicken embryos.     

Etude de la migration des cellules somitiques dans le mésoderme somatopleural de l'ébauche de l'aile

Summary In chick embryos, observations were made on serial semithin transverse sections of the wing level. In addition homo- or heterotopic replacements of the wing or leg somitic mesoderm by labelled somitic or nonsomitic mesoderm were made in 2-to 2.5-day embryos. The nuclear label used was either natural (quail donor embryos in heterotopic transplantations) or isotopic (chick donors labelled with tritiated thymidine).Histological examination revealed that the first somitic cells to leave somite 15 apparently did so at the 20 to 22 somite stage, while the last ones to leave somite 20 apparently did so shortly before the 36 somite stage.Transplantation experiments with labelled donor cells revealed the routes of migratory somitic cells and the time-course of their invasion into the outgrowing limb bud (non-somitic graft cells did not noticeably invade the limb anlage). They showed furthermore that the somitic mesoderm is not regionalized with respect to its limb myogenic properties.These results are compared with those obtained in other classes of vertebrates.

Ce travail a été subventionné en partie par la D.G.R.S.T. et le C.N.R.S.     

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.     

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.     

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.     

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)     

Adenovirus-mediated gene transfer as a tool to study angiogenesis in the chick embryo

Abstract. An adenoviral construct encoding a nuclear-localized beta-galactosidase marker protein was injected into the heart of chick embryos at Hamburger-Hamilton (HH) stage 14-15 (approximately 52-56 h of incubation). Reporter gene expression was determined 48-54 h after injection. Efficient gene transfer into endothelial cells (ECs) of intraembryonic and yolk sac vessels was observed. ECs of vessels in the head region, which undergo massive expansion around the time of injection, were efficiently labeled. However, limb bud vasculature, which starts to develop around stage 16 (HH), carried scarce (wing bud) or no (leg bud) lacZ marker. In contrast, ECs of the allantois, a structure that develops even later (around stage HH 18), expressed lacZ reporter. This observation suggests that EC precursors infected at an earlier time migrated into the allantois. A few non-endothelial cell types were also labeled by the reporter. These results suggest that adenovirus-mediated gene transfer provides a powerful tool to study angiogenesis in the developing chick embryo.