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Biomedical subjects

L Wolpert

Publications and source records attributed to L Wolpert.

At least 19 recordsLinked to original sources
PubMed1992

Development of left/right handedness in the chick heart.

The chick heart tube develops from the fusion of the right and left areas of precardiac mesoderm and in almost all cases loops to the embryo's right-hand side. We have investigated whether any intrinsic difference exists in the right and left areas of precardiac mesoderm, that influences the direction of looping of the heart tube. Chick embryos incubated to stages 4,5 and 6 were cultured by the New method. Areas of precardiac mesoderm were exchanged between donor and host embryos of the same stage and different stages to form control, double-right and double-left sided embryos. Overall, double-right sided embryos formed many more left-hand loops than double-left sided embryos. At stages 4 and 5 a small percentage of double-right embryos formed left-hand loops (13%) whereas at stage 6 almost 50% of hearts had left-hand loops. Control embryos formed right-hand loops in 97% of cases. The stability of right-hand heart looping by double-left sided embryos, may be related to the process of 'conversion', whereas the direction of looping by double-right sided embryos has become randomised. There is some indication that an intrinsic change occurred in the precardiac mesoderm between stages 5 and 6 that later influenced the direction of looping of the heart tube. The direction of body turning is suggested to be linked to the direction of heart looping.

Animals↗
PubMed1992

Gastrulation and the evolution of development.

The original eukaryotic cell may have possessed the key processes necessary for metazoan development--cell differentiation, patterning and motility--and these are present in the cell cycle. Protozoa also possess key patterning processes. It remains a problem as to why there should be two main modes of development--one based on asymmetric cell division and the other on cellular interactions. The latter may be related to asexual reproduction. The morphogenetic movements of gastrulation--as distinct from specifying the body plan--are highly conserved in a wide variety of organisms. This may reflect the requirement for patterning being specified in two dimensions, sheets of cells, and a third dimension being created by cell infolding. The origin of the gastrula can be accounted for in terms of Haeckel's gastrea theory--an early metazoan resembling the gastrula. Gastrulation in Cnidaria may resemble the primitive condition but there is nevertheless considerable diversity. While this may reflect, for example, yolkiness, it seems that there is little selection on developmental processes other than for reliability. Thus it is possible that the embryo is privileged with respect to selection and this may help account for the evolution of novel processes like the origin of the neural crest. Reliability is the key demand made on development. This may be provided by apparent redundancy. Since many developmental processes involve switches and spatial patterning reliability is provided by parallel buffering mechanisms and not by negative feedback.

Animals↗
PubMed1991

Expression of the homeobox Hox-4 genes and the specification of position in chick wing development.

The chicken Hox-4 homeogenes, like those of the mouse, are coordinately expressed in partially overlapping domains during wing development. Local application of retinoic acid, a putative endogenous morphogen, induces de novo transcription of Hox-4 genes. The mirror-image patterns of Hox-4 gene expression, which are obtained in this way, correlate with the subsequent development of mirror-image patterns of digits. Hox-4 genes probably encode positional information.

Amino Acid Sequence↗
PubMed1991

Development of handed body asymmetry in mammals.

We have proposed a three step model for the specification of left-right in mammalian embryos. The fundamental assumption is that handedness is imparted by an asymmetrical molecule. Conversion of molecular asymmetry to the cellular level gives a property to one side of the embryo to bias an otherwise random generation of an asymmetrical gradient which can be interpreted by developing organs. Rat embryos, treated at discrete stages, show a window of sensitivity for disruption of handedness, which may reflect the time of conversion/biasing. Heat shock and several chemicals cause left-right inversion in up to 50% of embryos exposed during neural groove formation. Earlier stages are less sensitive; no treatment begun after foregut pocket formation influences asymmetry. Evidence for cellular interactions in left-right specification comes from the apparent rescue of iv/iv mutant embryos in chimeras. We are looking for molecular left-right disparity before morphological asymmetry but detect no differences in two-dimensional protein profiles. Using an indirect measure, we find a right-left gradient of tissue oxygen in embryos at the 20-30 somite stage. This may reflect asymmetrical vasculature, as we have suggested to explain drug-induced asymmetrical limb malformations.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1990

Covalent modification of proteins as a threshold mechanism in development.

Thresholds are a central but somewhat neglected aspect of cellular processes in development. An analysis has been made of the conditions in which different thresholds can be generated in the covalent modification of a number of target proteins when the concentration of an effector is continuously increased. It is assumed that the effector, which could represent a morphogen, activates, for example, kinases that phosphorylate the proteins. Thresholds are found when the modifying enzymes are saturated by their protein substrates, i.e. in conditions of zero-order ultrasensitivity (Goldbeter, A. & Koshland, D. E. 1981. An amplified sensitivity arising from covalent modification in biological systems. Proc. natn. Acad. Sci. U.S.A. 78, 6840-6844). Sequential thresholds can be generated when the kinase/phosphatase pairs differ either in the ratio of maximum modification rates or in the affinity of the effector for each kinase.

Animals↗
PubMed1990

Expression of a homeobox gene in the chick wing bud following application of retinoic acid and grafts of polarizing region tissue.

Homeobox gene XlHbox 1 is expressed in a mesodermal gradient in vertebrate forelimbs with maximal expression anteriorly and proximally and may encode positional values. In chick wing buds, anterior cells can be reprogrammed to form posterior structures by grafts of polarizing region tissue and by beads soaked in retinoic acid (RA), which is a good candidate for an endogenous morphogen. Applications of RA anteriorly or at the bud apex, treatments which produce duplicated digits or truncations respectively, substantially increase the extent of mesodermal XlHbox 1 expression. Polarizing region grafts that also produce additional digits lead to a moderate increase. The effects of RA application and the behaviour of transplanted tissue show that only anterior cells are competent to express XlHbox 1 and that expression is cell autonomous. Ectodermal expression in wing buds is enhanced by RA but not by polarizing region grafts and ectoderm/mesoderm recombinations show that the mesoderm is irreversibly affected. The changes in mesodermal expression do not fit the predictions of the simple model that XlHbox 1 encodes anterior positional values but are correlated with a series of novel malformations of the shoulder girdle which, in normal wing buds, is derived from cells expressing XlHbox 1.

Animals↗
PubMed1990

Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation.

In insulin containing defined medium TGF-beta 1, TGF-beta 2, and bFGF all stimulate chondrogenic differentiation in high-density micromass cultures of distal limb bud mesenchyme cells of chick embryos. In addition bFGF inhibits myogenic differentiation, while TGF-beta 1 and TGF-beta 2 appear to have no effect. TGF-beta 1 and bFGF together act additively to enhance chondrogenesis, while TGF-beta blocks the bFGF inhibitory action on myoblasts, thus allowing them to differentiate. In the absence of insulin, the inhibitory effect of bFGF on muscle cell differentiation is reduced; cartilage differentiation in the presence of the above growth factors is also slightly reduced.

Animals↗
PubMed1990

The development of handedness in left/right asymmetry.

The development of handed asymmetry requires a special mechanism for consistently specifying a difference between left and right sides. This is to be distinguished from both random asymmetry, and from those left/right differences that are mirror symmetrical. We propose a model for the development of handedness in bilateral animals, comprising three components. (i) A process termed conversion, in which a molecular handedness is converted into handedness at the cellular level. A specific model for this process is put forward, based on cell polarity and transport of cellular constituents by a handed molecule. (ii) A mechanism for random generation of asymmetry, which could involve a reaction-diffusion process, so that the concentration of a molecule is higher on one side than the other. The handedness generated by conversion could consistently bias this mechanism to one side. (iii) A tissue-specific interpretation process which responds to the difference between the two sides, and results in the development of different structures on the left and right. There could be direct genetic control of the direction of handedness in this model, most probably through the conversion process. Experimental evidence for the model is considered, particularly the iv mutation in the mouse, which appears to result in loss-of-function in biasing, and so asymmetry is random. The model can explain the abnormal development of handedness observed in bisected embryos of some mammalian, amphibian and sub-vertebrate species. Spiral asymmetry, as seen in spiral cleavage and in ciliates, involves only conversion of molecular asymmetry to the cellular and multicellular level, with no separate interpretation step.

Animals↗
PubMed1990

Double anterior chick limb buds and models for cartilage rudiment specification.

Most models for the specification of the skeletal elements in the developing limb bud are based on a chemical specification well before overt cartilage differentiation. By contrast, a physico-mechanical model proposes that the process of condensation--an early feature of cartilage differentiation--is itself the basis for patterning the elements. The models thus make quite different predictions as to when the rudiments are specified. Double anterior limb buds have been constructed at stages earlier than condensation, with the expectation that, if specification of the humerus occurs before cartilage condensation, then limbs containing two humeri should develop, since the presumptive humerus lies largely in the anterior region. The development of anterior and posterior parts, on their own, was in general, consistent with the fate map; both developed a humerus that was thinner than normal. Double anterior limbs developed two humeri in 28% of cases and a much thicker humerus in 39%. These results strongly support models based on an early specification of limb rudiments and cannot be accounted for by the physical model. Double anterior limbs in which the two parts were from different stages, developed such that a digit 3 could lie adjacent to the radius, giving further striking evidence for early specification and local autonomy of development.

Animals↗
PubMed1990

The development of handed asymmetry in aggregation chimeras of situs inversus mutant and wild-type mouse embryo.

Mutant iv/iv mice develop as if they have no sense of left and right, so the development of asymmetry is random: half normal, half as a mirror-image of normal, situs inversus. We have made aggregation chimeras of 8-cell stage iv/iv and +/+ embryos, transferred them into pseudopregnant mice, and examined their phenotype on day 10 of gestation. The contribution of mutant and wild-type cells to tissues of the embryo was estimated by strain-specific isozyme (GPI-1) analysis. We have also performed reciprocal embryo transfers, iv/iv blastocysts into +/+ mice, and vice versa. These transfers show that the development of handed asymmetry is determined by embryonic genotype, and is unaffected by the maternal environment (at least after day 3), or by the procedures of embryo collection, culture and transfer. Our observations on the development of 21 viable chimeric embryos show that neither iv/iv nor +/+ cells are dominant. All embryos (12) with less than 50% contribution of iv/iv cells to the heart developed with normal situs. Of 9 embryos with greater than 50% iv/iv cells, only 2 developed with inverted situs. These findings suggests that there was partial 'rescue' of embryos by some influence of normal over mutant cells. However, we cannot, statistically, exclude an alternative interpretation that cells are behaving autonomously. Interestingly, the embryos that developed with inverted situs were unique in having greater than two thirds contribution of iv/iv cells to both the heart and the visceral yolk-sac.

Animals↗
PubMed1990

Signals in limb development: STOP, GO, STAY and POSITION.

Cell-to-cell interactions in early limb development are considered within the framework of the extracellular signals STOP, GO, STAY and POSITION, a classification which emphasises that the signals are elective rather than instructive, and that complexity arises from cells' response. Patterning in the limb is analysed in terms of signals that specify positional values along the anteroposterior axes, and retinoic acid is thought to be a positional morphogen. There is however, evidence for patterning which does not depend on a positional signal. In the early bud the mesenchyme gives POSITION signals to the apical ridge, which in turn provides a STAY signal to the mesenchyme in the progress zone. Non-ridge ectoderm produces a STOP signal with respect to cartilage differentiation. The pattern of cartilage differentiation is specified well before cartilage condensation. Growth factors affect both cartilage and muscle differentiation in culture. Pigment patterns result from feather germs providing STOP or GO signals to the melanoblasts which enter all feather germs. The pathways for the cell-to-cell signals are not known but may involve gap junctions.

Animals↗
PubMed1989

Ectodermal inhibition of cartilage differentiation in micromass culture of chick limb bud mesenchyme in relation to gene expression and cell shape.

Ectoderm inhibits the formation of cartilage by chick wing bud mesenchyme in micromass culture. This suggests that the pattern of cartilage formation in the limb bud may result from a restriction of cartilage cell differentiation to the limb bud core as cells leave the progress zone. We have used in situ hybridization to investigate whether ectodermal inhibition in micromass culture occurs at the level of gene transcription. We found that ectoderm completely inhibited the accumulation of cartilage-specific type II collagen transcripts in the mesenchyme cells, whilst the level of type I collagen transcripts was unaffected. Morphometric analysis of electron micrographs revealed that inhibition of chondrogenesis in micromass culture was not preceded by cell flattening. In fact, a rounded cell shape was found not to be a prerequisite for cartilage cell differentiation in micromass.

Animals↗
PubMed1989

The development of asymmetry: the sidedness of drug-induced limb abnormalities is reversed in situs inversus mice.

We are studying the development of handedness, in particular the relationships between handed structures with bilateral symmetry, for example the limbs, and those with lateral asymmetry, such as the heart, lungs and gut. Asymmetric (unilateral) developmental limb abnormalities can be induced by chemical treatment of mouse embryos, either in utero by acetazolamide, or in culture by misonidazole. We have examined these effects in mice homozygous for the iv gene. The development of bilateral symmetry in iv/iv mice is normal, but the control of asymmetry appears to be random, that is 50% develop normally (situs solitus), 50% with laterally inverted viscera (situs inversus). We find that the handedness of induced asymmetric limb defects is highly correlated with embryonic visceral situs. Right limb defects are induced in situs solitus embryos, left-sided defects in situs inversus. This suggests that the mechanism of induction of asymmetric defects is not related to any intrinsic difference between the development of left and right limbs, but is connected to visceral asymmetry. In addition, the high correlation of limb defects with situs was observed in culture as well as in utero suggesting that the maternal environment plays no role in the development of asymmetry.

Acetazolamide↗
PubMed1989

Positional information revisited.

Positional information has been suggested to play a central role in pattern formation during development. The strong version of positional information states that there is a cell parameter, positional value, which is related to position as in a coordinate system and which determines cell differentiation. A weaker version merely emphasises position as a key determinant in cell development and differentiation. There is evidence for boundaries and orthogonal axes playing an important role in positional systems. A positional signal is distinguished from an inductive interaction because the former specifies multiple states, confers polarity, and can act over a long range. A gradient in a diffusible morphogen is just one way of specifying position. There is now good evidence in several systems for substances which may be the morphogen for positional signalling. The product of the bicoid gene in early Drosophila development is the best prospect. Retinoic acid is unique in its ability to alter positional value and may also be a morphogen. The best evidence for positional value, a concept fundamental to positional information, remains a biological assay based on grafting. The idea of positional value uncouples differentiation and position, and allows considerable freedom for patterning. It is not clear whether positional value or differentiation involves a combinatorial mechanism. Interpretation of positional information remains a central problem. There is good evidence that cells can respond differentially to less than a two-fold change in concentration of a chemical signal. It may be that interpretation involves listing the sites at which a particular class of cell differentiation will occur. The problem is made less severe when blocks of cells are specified together as in mechanisms based on an isomorphic prepattern. Isomorphic prepatterns could establish repeated structures which are equivalent and which are then made non-equivalent by positional information. This would enable local differences to develop. The combination of these two mechanisms may be wide-spread. There is evidence that positional signals within a single animal and in related animals are conserved. It is not clear just how wide this conservation is, but it is at phylotypic stages, rather than in eggs, that similarity might be expected. It is nevertheless impressive that the polar coordinate model can be applied to regulation in systems as diverse as insects, vertebrates and protozoa. The molecular basis of positional signalling is just becoming accessible; the molecular basis of positional value is still awaited. A brief personal history of positional information is provided in an appendix.

Animals↗