Diffusible Morphogen Research Articles

Positional signalling along the anteroposterior axis of the chick wing. The effect of multiple polarizing region grafts

We have proposed that positional information along the anteroposterior axis is specified by a signal from the polarizing region and that position may be specified by the concentration of a diffusible morphogen. While this model can account for a variety of results it is now clear that a model based on intercalation by growth of positional values can do the same. The distinction between the two models lies in whether a grafted polarizing region can alter existing positional values and in the distance over which it exerts its influence. The two models make different predictions as to the effect of grafting two polarizing regions. The intercalation model predicts that this effect will be the sum of two single grafts, whereas the morphogen model predicts different results depending on how close together the two polarizing regions are placed. The pattern of digits following grafts of two polarizing regions show that it is sensitive to the distance between the grafts and consistent with a model based on long-range interaction, such as a diffusible morphogen.

Pattern formation along the anteroposterior axis of the chick wing: the increase in width following a polarizing region graft and the effect of X-irradiation

A study is made of the widening of the chick limb bud that occurs after a graft of an additional polarizing region. Such buds are about 50% wider than controls, after 36 h. By contrast, growth along the proximodistal axis in unaffected. This widening is reduced by treating the host embryo with 10 Gy X-irradiation and the altered pattern of digits is consistent with a diffusible morphogen model for the specification of positional information along the anteroposterior axis.

Positional signal transmission in the developing chick limb.

It has been suggested that the pattern along the antero-posterior axis of the embryonic developing chick limb arises from a gradient of diffusible morphogen produced by a special region of cells on the posterior margin of the limb bud, called the polarizing region. Grafts of posterior polarizing-region tissue to an anterior site in an embryonic chick wing bud result in mirror-image duplication of the limb bud; typically three extra wing digits are produced (Fig. 1f, g). Direct evidence for a long-range signal has, however, been lacking, and recently it has been suggested that chick wing development might better be understood as resulting only from local, neighbour-neighbour-like interactions of the sort postulated to occur during regeneration of insect and amphibian limbs and insect imaginal disks. As we report here, operations were performed in which chick wing buds were made host to grafts of leg bud responding tissue adjacent (posteriorly) to a grafted polarizing region (Fig. 2). The results showed that the pattern signal from the polarizing region can be transmitted through the leg tissue over distances of tens of cell diameters.

Wing buds with three ZPAs.

It has been hypothesized that a diffusible morphogen emanates from the ZPA of the embryonic chick limb and that cells can have their presumptive fate respecified to form more posterior structures when exposed to a higher concentration of morphogen. Attempts to raise the concentration of morphogen by grafting two additional ZPAs into a host wing bud failed. Instead, the greater the positional disparity between the donor and host tissue at the graft sites, the greater the number of extra structures that formed.

The time required for positional signalling in the chick wing bud

An estimate of the time required for positional signalling in the chick wing bud was obtained by grafting irradiated quail polarizing regions to host wing buds and removing them at various times. Such polarizing regions must be present for at least 15 h to induce additional structures in the host wings. This results is discussed in terms of a diffusible morphogen model.

Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue

The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host's limb bud or reversed to that of the host's. Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen.

Segmental gradients specifying polarity and pattern in the wax moth, Galleria mellonella: The posterior margin as the source of a diffusible morphogen

The basis for our experiments was a report by Piepho and Hintze-Podufal 1971 about the ability of the segmental posterior margin (PM) to induce alteration of polarity and pattern in abdominal segments of the wax moth Galleria mellonella. We planned to determine (1) whether the occurrence of ectopic PM on the host segment is due to integration of donor PM cells into the host epidermis and, if not, (2) what is the communication process leading to these developmental changes. Various intersegmental implantation and grafting experiments were carried out in late last instar larvae and the response of the host cells was determined by noting changes in orientation and morphology of the scales and sockets in the adult segment. A piece of the 5th PM implanted into the middle of the 7th segment near the surface, with the implant and host epidermis facing each other, induced overlying host cells to form 7th PM-type scales. This pattern transformation is not due to integration of donor PM cells into the host epidermis since grafted PM cells always retain their segmental identity. The implanted PM may cause reorientation of overlying host scales in a centripetal manner with or without accompanying pattern change. Both polarity and pattern changes can occur even when a nucleopore filter (0.03 μm) or a thin slice of agar is interposed between implant and host epidermis. From these and two cases of cell-free induction, we conclude that the PM is probably the source of a diffusible morphogen which influences polarity and pattern. The morphogen appears to be produced only during early stages of the larval-pupal molt and the host cells are sensitive to the factor only during critical periods in morphogenesis. The segmental anterior margin (AM) has an influence on polarity but appears to exert this effect only through cell contact. Our results are discussed in relation to current models of polarity and pattern regulation in the insect integument.

The zone of polarizing activity: evidence for a role in normal chick limb morphogenesis

When an impermeable barrier is placed so as to divide the early chick limb-bud into anterior and posterior parts then development continues only on one side of the barrier. The detailed results are inconsistent with mosaic development. They can readily be explained by supposing that pattern is specified by the concentration of a diffusible morphogen controlled by the zone of polarizing activity. A simulation of appropriate concentration profiles is presented and its relevance to similar experiments published elswhere is discussed. It seems probable that the zone of polarizing activity is active during normal development.

The position of regenerating cambia: auxin/sucrose ratio and the gradient induction hypothesis.

To account for the positions in which vascular cambia regenerate in wound callus, a gradient induction hypothesis was proposed in 1961 in terms of gradients in 'some factor as yet unknown'. It now seems likely that the gradient is based on morphogen diffusion between source and sink on opposite sides of existing cambia, with morphogen diffusing into the adjoining wound callus. It is specifically proposed that there are two morphogens, auxin diffusing centrifugally and sucrose diffusing centripetally. The cambium then regenerates along a path where the ratio of auxin to sucrose concentration is similar to that at the original cambium, and its orientation (as regards xylem and phloem formation) is determined by the direction of the gradient in this ratio. These proposals are supported by published evidence on auxin and sucrose concentration gradients across the cambium, and on their sources, movements, and known effects on vascular differentiation. Simulations of the proposed positional control system predict patterns of cambial regeneration and orientation corresponding to those observed in four different types of wound and graft.

Attenuation of positional signalling in the chick limb by high doses of gamma-radiation.

THE spatial pattern of cellular differentiation in the chick limb can be viewed in terms of positional information. For the antero–posterior axis of the wing, positional information seems to be specified by reference to a group of cells at the posterior margin of the limb bud—the zone of polarising activity (ZPA)1–3. By grafting an additional ZPA to different positions along the antero–posterior axis, we found that structures form according to their distance from the nearest ZPA. For instance, digit 4 forms nearest to the ZPA, then digit 3, then digit 2. We suggested that cells might measure distance by means of a diffusible morphogen produced by the ZPA, the concentration of which falls as distance from the ZPA increases. The responding cells are able to interpret the concentration of the morphogen, and so behave according to their position. If it is the concentration of morphogen that specifies which digit shall form, then lowering the concentration of morphogen adjacent to the ZPA should result in digit 3 being formed next to the ZPA instead of digit 4. If the concentration is lowered still further, digit 2, and finally no additional digit should form next to the ZPA. We have obtained such an attenuation of positional signalling by treating the ZPA with increasing doses of γ radiation.

Thresholds in development

The interpretation of gradients in positional information is considered in terms of thresholds in cell responses, giving rise to cell states which are discrete and persistent. Equilibrium models based on co-operative binding of control molecules do not show true thresholds of discontinuity, though with a very high degree of co-operativity they could mimic them; in any case they do not provide the cells with any memory of a transient signal. A simple kinetic model based upon positive feedback can account both for memory and for discontinuities in the pattern of cell states. The model is an example of a bistable control circuit, and transitions from one state to another may be brought about not only by morphogenetic signals, but also by disturbances in the parameters determining the kinetics of the system. This might explain some aspects of transdetermination in insects. An attempt is made to analyse the precision with which a spatial gradient of a diffusible morphogen could be interpreted by a kinetic threshold mechanism, in terms of the length of the field, the steepness of the concentration gradient, and the intrinsic random variability of cells. It is concluded that it would be possible to specify as many as 30 distinct cell states in a positional field 1 mm long with a concentration span of 10 3. Mechanisms for reducing the positional error are considered.

Pattern stability in the insect segment

1. The mechanisms of pattern reconstitution in the abdominal segment of insects were studied by transplantations between the wild type and a colour mutant of the bugDysdercus intermedius. In this species, anterior and posterior regions of the segment differ in pigmentation. Thus transplants are marked by donor genotype and by region-specific pigmentation. Moreover, the translucent cuticle allows direct and continuous observation of the behaviour of host and transplant cells. 2. Transplants rotated by 90° re-rotate so as to approach or even restore the original relations with their surroundings, whereas transplants completely surrounded by cells from another segment region tend to contract. Both observations indicate differences in adhesiveness between cells from different regions of a segment. 3. When transplants rotated by 180° or shifted to different antero-posterior levels in a segment cannot approach their original situations by rerotation, then conspicuous folds appear before moults in the regions where cells from different segment levels meet, and these regions become pigmented according to the levels which were lacking in between. This indicates that an intercalary regenerate is formed which eliminates the discontinuity in positional values created by transplantation. 4. The intercalary regenerate forms essentially from cells representing the more posterior segment level, irrespective of whether these are host or transplant cells. 5. It is suggested that pattern reconstitution in the abdominal segment can be explained in terms of cell behaviour, assuming cell sorting and intercalary regeneration. 6. This interpretation is discussed in the light of a current hypothesis which assumes that displaced cells are re-programmed in situ under the influence of a diffusible morphogen.

A model generating the pattern of cartilage skeletal elements in the embryonic chick limb

The development of the vertebrate limb involves the production of a specific external form arising from cell division and other growth processes at the cellular level, and the origin within it of specific patterns of tissues arising by cellular differentiation, of which the pattern of cartilages which pre-figure the limb skeleton is the most striking. In this paper we propose a model for the differentiation (or the preceding determination) process that, using only localized cell to cell interactions, can approximate the cartilage pattern in any limb shape. The model requires cells to modify their metabolism irreversibly at critical threshold levels of a diffusible morphogen which may be made or destroyed by these cells. Restrictions inherent in the successful development of a total limb pattern using this system lead to the prediction that the process is confined to a distal band which has no significant interaction with more proximal regions but within this band the characteristic features of the anterior-posterior axis of the limb develop without additional interactions. Cartilage elements are initiated as single “cells” and expand centrifugally to their final size; these elements developing sequentially along the anterior-posterior axis, showing a distinct polarity of size. The model also predicts that equivalent cartilage elements in all vertebrate limbs will be roughly the same relative size at determination, the extensive range of adult structures arising by differential growth and fusion, possibly controlled by global aspects of the model. It must be emphasized that this model only satisfactorily simulates the anterior-posterior patterning of cartilage elements, the disto-proximal pattern being externally imposed. The final cartilage pattern is shown to be a function of (1) the developing shape of the limb, (2) the position of an initiator region that starts the patterning process and (3) the rate of production of the diffusible morphogen. Using parameters selected with as much realism as possible the model gives a good approximation to the pattern of c cartilages found in the normal chick limb; modifying the shape of the limb to that of the talpid 3 mutant produces the characteristics features of the cartilage pattern found in that mutant and modifying the rate of morphogen production simulates patterns resembling those found in some ancestral vertebrate fossil forms.