Biomedical subjects

S V Bryant

Publications and source records attributed to S V Bryant.

At least 19 recordsLinked to original sources

PubMed1992

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)

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PubMed1992

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.

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PubMed1992

Retinoic acid, local cell-cell interactions, and pattern formation in vertebrate limbs.

Retinoic acid (RA), a derivative of vitamin A, has remarkable effects on developing and regenerating limbs. These effects include teratogenesis, arising from RA's ability to inhibit growth and pattern formation. They also include pattern duplication, arising as a result of the stimulation of additional growth and pattern formation. In this review we present evidence that the diverse effects of RA are consistent with a singular, underlying explanation. We propose that in all cases exogenously applied RA causes the positional information of pattern formation-competent cells to be reset to a value that is posterior-ventral-proximal with respect to the limb. The diversity of outcomes can be seen as a product of the mode of application of exogenous RA (global versus local) coupled with the unifying concept that growth and pattern formation in both limb development and limb regeneration are controlled by local cell-cell interactions, as formulated in the polar coordinate model. We explore the possibility that the major role of endogenous RA in limb development is in the establishment of the limb field rather than as a diffusible morphogen that specifies graded positional information across the limb as previously proposed. Finally, we interpret the results of the recent finding that RA can turn tail regenerates into limbs, as evidence that intercalary interactions may also be involved in the formation of the primary body axis.

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PubMed1991

Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud.

In recent years there has been considerable interest in the role of retinoic acid (RA) in vertebrate-limb pattern formation. When RA is applied to the anterior of the chick wing bud, a mirror-image duplication of the limb pattern develops that is identical to the pattern resulting from grafts of posterior tissue (zone of polarizing activity, or ZPA). It has been proposed that position along the anterior-posterior axis in the chick limb is specified by a gradient of a diffusible factor produced by the ZPA. The ZPA-mimicking action of RA has led to the hypothesis that exogenously applied RA acts by providing graded spatial information across the anterior-posterior limb axis. An alternative interpretation is that RA changes anterior cells into ZPA cells, which in turn provide the actual pattern-duplicating stimulus; there is already some preliminary evidence that this occurs. A hybrid interpretation has also been suggested whereby ZPA cells are formed in response to RA exposure and then begin to release retinoids that act as graded spatial cues. We have used a functional assay to test anterior chick wing-bud cells for ZPA activity after exposure to RA. The results of our studies indicate that the action of RA is to change anterior cells into ZPA cells. Further, our results indicate that it is unlikely that RA-treated anterior cells then begin producing RA in such a way as to provide a graded positional signal.

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PubMed1991

Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotls and binds specifically to blastema tissues.

The growth of regenerating limbs of amphibians depends upon proliferation of the blastema cells that accumulate beneath the epidermal cap. The epidermal cap is known to be mitogenic for the blastema cells. We have extracted a mitogenic activity from both the mesenchymal and epidermal (epidermal cap) components of cone stage blastemas which is retained on heparin-Sepharose and elutes with 1.15 M NaCl. This fraction stimulates neurite outgrowth of PC12 cells and [3H]thymidine incorporation into CCL 39 cells and is potentiated by heparin. The 2 M fraction was inactive. The heparin-Sepharose-purified growth factor cross-reacts with bovine acidic FGF polyclonal antibodies and shows a Mr of 16,000 on Western blots. Blastema membranes contain specific high affinity binding sites (Kd = 25 pM; capacity = 30 fmole/mg protein) and low affinity binding sites (Kd = 18 nM; capacity = 30 pmole/mg protein) for aFGF as revealed by Scatchard analysis. 125I-aFGF which is bound specifically by both the epidermal cap and mesenchyme of blastema frozen sections is displaced by an excess of unlabeled factor and inhibited by heparin. Heparinase treatment and 2 M NaCl washing which decreased the binding was fourfold more efficient for epidermal cap than for mesenchyme suggesting the presence of high affinity receptors in the latter tissue. The presence of aFGF (or a closely related molecule) in blastemas is consistent with our earlier results that showed stimulation of proliferation of cultured blastema cells by acidic or basic FGF or heparin alone. These results suggest the possibility that aFGF is stored in the epidermal cap during limb regeneration and that it stimulates the proliferation of the underlaying mesenchyme.

Ambystoma↗

PubMed1991

Effects of localized application of transforming growth factor beta 1 on developing chick limbs.

The effects of exogenous transforming growth factor beta (TGF-beta) on chick limb development in vivo were studied by implanting carriers of TGF-beta 1 into developing wing buds. Agarose beads were soaked in solutions containing TGF-beta 1 and implanted into wing buds at stages 18 to 27. Localized application of TGF-beta 1 to distal regions of the wing bud caused specific skeletal elements in the limb to be reduced or absent. The particular proximal-distal limb element affected depended on the stage at which the bead was implanted. Position of the bead in the anterior-posterior axis also influenced the pattern of affected structures. Experiments in which TGF-beta 1 beads were implanted and then removed at 24- and 48-hr intervals indicate that there are specific periods during which a skeletal element appears to be sensitive to the effects of exogenous TGF-beta 1. In a few cases, beads placed in proximal positions in later staged limbs resulted in formation of ectopic cartilage near the bead. These results suggest that exposure to exogenous TGF-beta 1 in vivo influences the development of skeletal elements in the chick limb in a stage- and position-dependent manner.

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PubMed1991

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.

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PubMed1989

A staging system for mouse limb development.

A series of 15 stages of development for the mouse limb bud have been defined, spanning the time from the first appearance of the limb bud to the completion of limb outgrowth. The stages are based on changes in the morphology of the limb in living preparations. The development and regression of the apical ectodermal ridge (AER) as well as the development of the skeletal structures are also described. This staging system has been developed in response to the need to standardize in situ experimental analyses of the mouse limb bud. Comparable stages of the commonly used chick wing and mouse whole embryo systems are presented.

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PubMed1989

Mammalian limb bud development: in situ fate maps of early hindlimb buds.

Fate maps of the developing mouse hindlimb bud have been constructed for the first time using exo utero surgical techniques and carbon particle injections. Such fate maps demonstrate that the limb develops in a proximal to distal manner as a result of distal expansion. The anterior-posterior extent of the limb bud develops asymmetrically with the posterior half giving rise to slightly more of the digit pattern (digits 3-5) than the anterior half (digits 1 and 2). We found no evidence for the occurrence of extensive cellular rearrangements during limb development, and the free limb bud appears to give rise to only zeugo- and autopodial elements with the stylopod arising in the body wall proximal to the bud. These results are consistent with our current understanding of limb development in lower vertebrates and also provide detailed information that will be useful for future limb studies in mammals.

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PubMed1989

Evidence for regulation following amputation and tissue grafting in the developing mouse limb.

Procedures are now available to experimentally manipulate postimplantation mouse embryos in situ and allow development to continue into postnatal life (Muneoka, K., N. Wanek, and S.V. Bryant (1986) J. Exp. Zool., 239:289-293). We have investigated the ability of the well-formed hindlimb bud to regulate following two experimental operations: amputation and wedge grafts designed to confront anterior and posterior cells. After comparing the resultant limbs with the fate maps of the relevant stages, we conclude that the developing hindlimb bud at stage 7/8 (equivalent to stage 27/28 of the chick) is capable of partial regeneration of the peripheral digits following amputation and capable of supernumerary digit tip formation after grafting of a wedge of anterior tissue to a posterior position.

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PubMed1989

Organization of positional information in the axolotl limb.

We have used the phenomenon of position-dependent growth stimulation, brought about by the confrontation of cells with dissimilar positional values, to reveal the organization of positional information in the center of the upper and lower arms of axolotls. When either humerus or radius was transplanted into either dorsal or posterior positions, extra growth leading to the formation of supernumerary digits occurred following amputation through the graft. However, transplants of humerus or radius into anterior or ventral positions did not lead to the formation of any additional digits. The ulna by contrast was capable of stimulating supernumerary digit formation when transplanted into anterior, posterior, dorsal, or ventral positions. We interpret these results to indicate that the humerus and radius are surrounded by symmetrically arranged anterior and ventral positional values, whereas the ulna is surrounded by a complete asymmetrical set of angular positional values. We use our proposed arrangement for the positional information in the limb center to explain a number of previous experimental findings. In addition, we provide an explanation, in terms of the underlying positional information, for the structural and developmental relationships between the different skeletal elements of the vertebrate limb, and in particular for the anatomical pattern known as Gregory's pyramid.

Ambystoma mexicanum↗

PubMed1989

Compatible limb patterning mechanisms in urodeles and anurans.

We have experimentally tested the similarity of limb pattern-forming mechanisms in urodeles and anurans. To determine whether the mechanisms of limb outgrowth are equivalent, we compared the results of two kinds of reciprocal limb bud grafts between Xenopus and axolotls: contralateral grafts to confront anterior and posterior positions of graft and host, and ipsilateral grafts to align equivalent circumferential positions. Axolotl limb buds grafted to Xenopus hosts are immunologically rejected at a relatively early stage. Prior to rejection, however, experimental (but not control) grafts form supernumerary digits. Xenopus limb buds grafted to axolotl hosts are not rejected within the time frame of the experiment and therefore can be used to test the ability of frog cells to elicit responses from axolotl tissue that are similar to those that are elicited by axolotl tissue itself. When Xenopus buds were grafted to axolotl limb stumps so as to align circumferential positions, the majority of limbs did not form any supernumerary digits. However, in experimental grafts, where anterior and posterior of host and graft were misaligned, supernumerary digits formed at positional discontinuities. These results suggest that Xenopus/axolotl cell interactions result in responses that are similar to axolotl/axolotl cell interactions. Furthermore, axolotl and Xenopus cells can cooperate to build recognizable skeletal elements, despite large differences in cell size and growth rate between the two species. We infer from these results that urodeles and anurans share the same limb pattern-forming mechanisms, including compatible positional signals that allow appropriate localized cellular interactions between the two species. Our results suggest an approach for understanding homology of the tetrapod limb based on experimental cellular interactions.

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PubMed1988

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus.

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.

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PubMed1986

Mouse embryos develop normally exo utero.

Surgical procedures are described which permit direct experimental access to postimplantation mouse embryos. These procedures arose from our finding that development proceeds normally to term after embryos have been released from the confines of the uterus, while remaining attached to it via the placenta. Embryos continue to develop exo utero, within the abdominal cavity of the mother, and are capable of surviving a variety of different surgical manipulations. This finding opens the way for experimental analyses of mouse development in vivo.

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PubMed1986

Intrinsic control of regenerative loss in Xenopus laevis limbs.

The regenerative capability of Xenopus laevis hindlimbs was studied at different developmental stages. Three types of surgical deletion of the autopod were performed: simple amputations, 3-digit wedge-shaped deletions and 1-digit wedge-shaped deletions. The frequency of regenerative response and the digital patterns of the regenerates were analyzed. Regenerative capacity declines with developmental stage for all three types of deletions. For simple amputations this decline occurs in an orderly manner with the failure of anterior digits to regenerate at progressively later stages. A comparison between 1-digit and 3-digit deletions shows that for all stages of development, 3-digit deletions regenerate better than one-digit deletions. These data indicate that the amount of tissue removed is directly related to whether a regenerative response is observed. At any given stage, larger deletions are more likely to regenerate than smaller ones. These results are discussed with regard to the mechanisms by which growth during regeneration is controlled.

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PubMed1986

The migration of dermal cells during blastema formation in axolotls.

Using the diploid/triploid cell marker in the axolotl (Ambystoma mexicanum) we have examined the movement of cells from the dermis into the early limb blastema. Cells of dermal origin begin to migrate beneath the wound epithelium at about 5 days postamputation, and by 10 days they are widely distributed across the amputation surface. By 15 days, a dense accumulation of blastema cells is present beneath the apical cap, and these cells are preferentially oriented in a circumferential direction. These results are discussed in relation to previous studies showing that the progeny of dermal cells become widely distributed during regeneration, and that cells of dermal origin are a major source of blastema cells. The results are also discussed in relation to ideas about how growth and patterning of the new appendage occur.

Ambystoma mexicanum↗

PubMed1986

Cellular contribution from dermis and cartilage to the regenerating limb blastema in axolotls.

Using the triploid/diploid cell marker in the axolotl, Ambystoma mexicanum, we have analyzed the extent to which cells derived from the dermis and the skeleton contribute to the regenerating limb blastema. We found that dermal cells contribute 43% of the blastemal cell population whereas cells derived from skeletal tissue contribute only 2%. When compared to the availability of cells at the plane of amputation, dermal cells overcontribute by greater than twofold whereas skeletal cells undercontribute by several-fold. These data correlate with the effects that these two tissues have on the formation of the limb pattern during regeneration; dermis has a dramatic influence on pattern and skeletal tissue has virtually no effect. It is suggested that the fibroblasts present in the dermis and in other parts of the limb form virtually all of the mesodermal tissues in the regenerate with the exception of the muscle.

Ambystoma mexicanum↗

PubMed1986

Pattern discontinuity, polarity and directional intercalation in axolotl limbs.

Axolotl limb stumps with dorsal-ventral confrontations between digits 2 and 3 but with a normal anterior-posterior pattern were created by grafting between contralateral limbs. Graft and host differed in ploidy to permit a determination of the origin of cells in the regenerated limb. After regeneration, limbs were analysed for skeletal and muscle patterns and for the distribution of marked cells in the regenerate. Regenerated limbs showed varying degrees of abnormality in their dorsal-ventral organization. Following regeneration, the original dorsal-ventral discontinuities were in some cases maintained and in others resolved. The maintenance or resolution of pattern discontinuities occurred in a position-dependent manner. Cell marker analysis indicates a relationship between the resolution of discontinuities and the extent to which cells become displaced across the original graft-host interface. These data lend support to the suggestion that circumferential intercalation is directionally biased.

Ambystoma↗