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.     

A gradient of gap junctional communication along the anterior-posterior axis of the developing chick limb bud.

A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20/21) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities.     

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.     

Position of origin of donor posterior chick wing bud tissue transplanted to an anterior host site determines the extra structures formed

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.     

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)     

Pattern Specification and Pattern Regulation in the Embryonic Chick Limb Bud

SYNOPSIS. The embryonic chick limb bud is a growing organ rudimentwhose undifferentiated cells give rise to a precise spatialpattern of differentiated structures. The establishment of positionalvalues of chick limb bud cells (pattern specification) and theresponse of limb bud cells with established positional valuesto experimental perturbations (pattern regulation) are the majortopics considered in this paper. The results of recent experimentswith developing chick limb buds analyzing pattern specificationand pattern regulation are presented. These studies with thechick limb are described in light of the postulates of a modelthat was originally formulated from experiments performed onregenerating amphibian and insect appendages.     

Behavior of cells in artificially made cell aggregates and tissue fragments after grafting to developing hind limb buds in Xenopus laevis

During vertebrate limb development, the limb bud grows along the proximo-distal (P-D) direction, with the cells changing their adhesiveness. To know whether the position-related differences in cell adhesiveness are actually utilized by morphogenesis to constitute limb structures, we grafted cell aggregates made of dissociated cells derived from different positions and stages of developing hind limb buds into developing hind limb buds and observed the behavior of the cells. Cell aggregates made of dissociated mesenchymal cells from two different origins were implanted in different positions and stages of limb buds or grafted on limb stumps made by cutting. The two grafted cell populations in the aggregate always sorted out from each other, but their patterning of sorting-out was quite different according to the transplanted regions. In summary, cells in the aggregate that have closer positional identity to the transplanted site were always situated at the boundary between host and donor cells. The pattern of sorting-out seemed to be determined by the relative adhesiveness of surrounding cells to the constituent cells of the aggregates. We also transplanted fragments dissected out from different regions along the P-D axis into st. 50 limb buds. The descendants of grafted cells moved distally to the region corresponding to their positional identity and participated in the formation of more distal structures from that point. These results suggest that the difference in cell adhesiveness may probably play a role in arranging cells along the P-D axis of a developing limb bud.     

Temporal and spatial expression of a position specific antigen, AV-1, in chick limb buds

In a previous study, we demonstrated the presence of a position-specific antigen (AV-1) in chick limb buds at an early developmental stage. Here, we reported the temporal and spatial expressions and the biochemical characterization of the AV-1 antigen. Indirect immunofluorescence staining and immunoblot analysis clearly showed that the AV-1 antigen is a glycoprotein that is localized on the plasma membrane and that it is expressed from stage 19 and highly expressed at stages 22-26 in some middle-distal to anterior-distal region of limb buds. In the wing bud, at stage 28, the AV-1 antigen was faintly detected in the restricted space between the precartilaginous regions of the radius and the ulna, and those of the metacarpals 2 and 3, but not those of the metacarpals 3 and 4. Such stage-specific and "position-specific" expressions of the AV-1 antigen in limb buds strongly suggest that the AV-1 antigen or cells containing it are involved in determination of the limb pattern formation.     

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.     

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)     

Regeneration potency of mouse limbs

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodismH (bpH) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bpH mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Position specific binding of a monoclonal antibody in chick limb buds

To analyze the molecular mechanism of the limb pattern formation, we have tried to make monoclonal antibodies against antigens from chick limb buds. We obtained one antibody named AV-1 which recognized a specific region of chick limb buds. AV-1 reacted with the distal portion of the anteroventral mesoderm of only developmentally early chick limb buds. Grafts of ZPA region tissue to an anterior site in an embryonic chick wing bud resulted in mirror-image dupliction of the AV-1 antigen region. These data show the possibility that this antigen plays some role in the limb pattern formation. This is the first evidence that a position specific substance really exists in developmentally early limb buds in which the pattern has been considered to be unspecified.     

Cooperative Activation of HoxD Homeobox Genes by Factors from the Polarizing Region and the Apical Ridge in Chick Limb Morphogenesis

When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted into the anterior margin of the chick limb bud, the expressions of the chick homeobox genes HoxD12 and D13 were induced prior to the formation of chick extra digits. This induction was observed in a restricted domain close to both the grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the distaloposterior region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, act cooperatively to provide positional information to induce the sequential expression of the HoxD genes.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Abstract. Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism^H ( bp ^H ) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bp ^H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Mouse limb bud cells respond to retinoic acid in vitro with reduced growth.

Retinoic acid (RA) has dramatic effects on the pattern of developing and regenerating vertebrate limbs. These effects are considered to result from RA-induced changes in the positional identity of limb cells, and involve the formation of extra structures. Whether the growth required to form the supernumerary parts of the pattern is a primary effect of RA treatment or a secondary effect that follows after a change in positional identity is not at present known. In this paper we have investigated the effects of RA treatment on the growth of cells from anterior and posterior halves of mouse limb buds in vitro. We observed that under our culture conditions, limb bud cells treated with 1 nM to 1 microM RA (0.3 ng/ml to 300 ng/ml) continue to grow but do so at a significantly slower rate than control cultures. There is a maximum inhibition of growth (50% of controls) between 10 nM and 100 nM RA, which corresponds to the measured range of concentrations of RA in vivo. Our observation of a significant decrease in growth rate over a wide range of RA concentrations is consistent with comparable reports of growth inhibition for a large number of other cell types in vitro as well as with the observation that exogenous RA inhibits blastemal growth in amphibians during the period of exposure to RA. We propose that the effects of RA on growth, either enhancement in vivo or reduction in vitro, can be seen as consequences of the ability of RA to alter positional identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ghox 4.7: a chick homeobox gene expressed primarily in limb buds with limb-type differences in expression.

Homeobox genes play a key role in specifying the segmented body plan of Drosophila, and recent work suggests that at least several homeobox genes may play a regulatory role during vertebrate limb morphogenesis. We have used degenerate oligonucleotide primers from highly conserved domains in the homeobox motif to amplify homeobox gene segments from chick embryo limb bud cDNAs using the polymerase chain reaction. Expression of a large number of homeobox genes (at least 17) is detected using this approach. One of these genes contains a novel homeobox loosely related to the Drosophila Abdominal B class, and was further analyzed by determining its complete coding sequence and evaluating its expression during embryogenesis by in situ hybridization. Based on sequence and expression patterns, we have designated this gene as Ghox 4.7 and believe that it is the chick homologue of the murine Hox 4.7 gene (formerly Hox 5.6). Ghox 4.7 is expressed primarily in limb buds during development and shows a striking spatial restriction to the posterior zone of the limb bud, suggesting a role in specifying anterior-posterior pattern formation. In chick, this gene also displays differences in expression between wing and leg buds, raising the possibility that it may participate in specifying limb-type identity.