Intercalary regeneration in legs of crayfish: Proximal segments

An arthropod leg represents a protuberance of the body segmental integument which bears distinctive markers in both the mediolateral and the anteroposterior axes. To clarify the biaxial organization of the body segmental morphogenetic field, and to study the relation among the whole-limb, limb segmental, and body segmental fields previously recognized in arthropods, we have grafted a proximal leg segment into the ventral midline in crayfish. After this operation the majority of animals regenerated a mirror-symmetric pair of supernumerary legs at the host site. Some of these legs had the most proximal segment, the coxa, partially fused to the adjacent body surface. Minority patterns of regeneration included one midline leg with a gill, three midline legs with a gill, and two normal legs with a third double-half leg. These results are compatible with the principle that intercalary regeneration restores the continuity of positional information.     

Discontinuities of pattern and rules for regeneration in limbs of crayfish

The most caudal limb in crayfish, the uropod, has two rami, the exopodite and the endopodite. Results of earlier experiments (J.E. Mittenthal et al. (1985) W. Roux's Arch. Dev. Biol. 194, 121-130) indicated that ramus morphogenetic fields in the two rami are equivalent and tandem. Thus the proximal (inner) junction of the rami, where intersegmental membrane separates them from each other and from the coxa, is analogous to a boundary between segments of the body or of a leg. In this region a discontinuity in the positional information carried by the ramus fields might occur. To characterize the morphogenetic fields in the region near this junction we have exchanged the medial and lateral margins of the two rami, performing the four possible grafting operations of this kind. While an experimentally generated discontinuity between the lateral margin of the exopodite and the medial margin of the endopodite (outer-to-outer junction) triggers intercalation of supernumerary rami, a discontinuity of pattern between the medial margin of the exopodite and the lateral margin of the endopodite (inner-to-inner junction) is stable despite the absence of intervening intersegmental membrane. Where intercalation does occur, it can proceed in either direction along the margin of a supernumerary ramus. These results suggest that there is no discontinuity of positional value at the boundary between the rami. The results of all of our experiments on the uropod indicate that a conjunction of separate proximodistal, dorsoventral, and mediolateral component fields may give positional information for generating the uropod. Intercalation restores the continuity of pattern in the proximodistal field. In the mediolateral field a discontinuity of pattern may result from a preferred polarity of intercalation: Outer cells may be competent to generate inner cells, but not vice versa. According to this hypothesis the two rami have tandem ramus fields but mirror-symmetric polarity of competence. Alternatively, intercalation may eliminate a mediolateral discontinuity only if the mismatch of mediolateral positional values at the discontinuity exceeds a threshold. The threshold criterion may be a weighted sum of limb field and ramus field positional values.     

A reaction-diffusion theory of morphogenesis with inherent pattern invariance under scale variations

In the framework of reaction-diffusion theory we deal with the problem of pattern regulation in morphogenesis. A generic model is proposed where the kinetic terms follow constraints imposed by scale invariance considerations. These constraints allow a class of kinetic schemes to be formulated so that, starting with an initially homogeneous morphogen distribution in the field, a stable gradient is established of the form: S(chi,L) = Lpf(chi/L). Here L is the length of the morphogenetic field, chi is the position variable and f(chi/L) is some monotonic function of the relative distance. With this distribution a scale invariant gradient can be constructed which leads to pattern regulation. A linear stability analysis of the model permits the definition of the parameter values enabling the system to abandon the homogeneous state spontaneously. Simulations of the evolution of the system towards its final stable state result in approximate pattern invariance for different field lengths. The accuracy of this invariance is in agreement with some recent quantitative experimental findings in both developing and regenerating systems.     

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 10³. Mechanisms for reducing the positional error are considered.     

Pattern Regulation of the Leg Disc of the Fleshfly, Sarcophaga peregeina

A fate map of the hind leg disc of Sarcophaga peregrina was constructed by examining the adult structures of implanted disc fragments. The locations of presumptive adult structures in the disc were similar to those of fore leg disc of Drosophila and Sarcophaga ruficornis . However, the concentric borderlines of the segments could not be ascertained in the present case.
Pattern regulation of disc fragments was studied by culturing them either in adult females for several days or for 3 days in mature larvae placed on wet condition. Cultured disc fragments regenerated or duplicated as in Drosophila , with some exceptions. For instance, the region with a high density of positional values, the upper medial quarter, of the fore leg disc of Drosophila was not found. A characteristic difference in the rate of regeneration or duplication was observed in the implanted fragments, when cultured in larvae or adult hosts. This variable pattern regulation in larval and adult hosts could be due to different compositions of the hemolymph in which would healing of the implanted disc fragments takes place.     

Shaping a Morphogen Gradient for Positional Precision

Morphogen gradients, which provide positional information to cells in a developing tissue, could in principle adopt any nonuniform profile. To our knowledge, how the profile of a morphogen gradient affects positional precision has not been well studied experimentally. Here, we compare the positional precision provided by the Drosophila morphogenetic protein Bicoid (Bcd) in wild-type (wt) embryos with embryos lacking an interacting cofactor. The Bcd gradient in the latter case exhibits decreased positional precision around mid-embryo compared with its wt counterpart. The domain boundary of Hunchback (Hb), a target activated by Bcd, becomes more variable in mutant embryos. By considering embryo-to-embryo, internal, and measurement fluctuations, we dissect mathematically the relevant sources of fluctuations that contribute to the error in positional information. Using this approach, we show that the defect in Hb boundary positioning in mutant embryos is directly reflective of an altered Bcd gradient profile with increasing flatness toward mid-embryo. Furthermore, we find that noise in the Bcd input signal is dominated by internal fluctuations but, due to time and spatial averaging, the spatial precision of the Hb boundary is primarily affected by embryo-to-embryo variations. Our results demonstrate that the positional information provided by the wt Bcd gradient profile is highly precise and necessary for patterning precision.     

Models for cell differentiation and generation of polarity in diffusion-governed morphogenetic fields

Models based on molecular mechanisms are presented for pattern formation in developing organisms. It is assumed that there exists a diffusion governed gradient in the morphogenetic field. It is shown that cellular differentiation and the subsequent pattern formation result from the interaction of the diffusing morphogen with the genetic regulatory mechanism of cells. In a second stage it is shown that starting from a homogeneous distribution of morphogen, polarity can be generated spontaneously in the morphogenetic field giving rise to the establishment of a gradient. The stability of these gradients is demonstrated. The onset of a morphogenetic gradient and pattern formation are combined in a single coherent model. Size invariance and its biological implications are discussed.     

Organization of the Morphogenetic Field in Regenerating Amphibian Limbs

The amphibian limb is an example of a secondary embryonic fieldthat can be reactivated during larval or adult life so thatamputated parts are regenerated. Two major questions are: (1)what is the origin of the morphogenetic field of the regenerationblastema, and (2) what is the nature of this field and how doesit specify the spatial pattern of blastemal redifferentiation?Evidence is analyzed here which leads to the following propositions:(1) the field is represented in latent form by properties ofthe mature limb cells, and these properties are activated andinherited by the blastemal cells after amputation and dedifferentiation.At the same time, the inherited field is sensitive to the maturestump tissues and its spatial organization can be altered bya stump pattern alien to the one from which it was derived.(2) The properties of the mesodermal limb tissues representpositional values that are arranged in gradients along the proximal-distal,anterior-posterior and dorsal-ventral axes. These propertiesallow dedifferentiated mesodermal cells to change their positionalvalue to any value between their original one in the limb andthe value of any neighboring cell after creation of a discontinuity.The direction of change is always from proximal to distal inthe PD axis; it is uncertain as to whether change can take placeonly centripetally or both centripetally and centrifugally alongthe AP and DV axes. (3) Epidermal cells have the same positionalvalue everywhere in the limb and act as the distal and circumferentialboundaries up to which the mesodermal cells may change theirpositional values. The proximal boundary is represented by thelevel-specific properties of the mesodermal cells at the maximumextent of distal to proximal dedifferentiation. Normal regenerationcan then be visualized as occurring in the following way. Whendeletions are made in the limb pattern, cells with widely differentpositional values are confronted. During regeneration, blastemacells increase in number and continually interact with theirneighbors to adjust their positional values within the boundariesuntil discontinuities are eliminated. The multiple limbs resultingfrom rearrangement of stump tissue patterns can also be accountedfor by using these propositions. It is suggested that positionalinformation is encoded on the cell surface and/or in the extracellularmatrix.     

Regeneration studies on a crayfish neuromuscular system. II. Effect of changing the nerve entry point into the muscle field on the gradient of innervation

The superficial flexor muscle of the crayfish is a neuromuscular system in which the neurons form position-dependent connectivity patterns with the muscle fibers. This system could be formed with the help of a single medial-to-lateral gradient during development that embodies positional information. To test this gradient hypothesis we changed the nerve's normal medial entry point into the muscle by transplanting it to the middle of the muscle sheet. When all the muscle fibers were present in the target area, most of the neurons studied passed through a stage during regeneration in which they showed preference for either medial or lateral synapse formation. Those neurons that in normal animals innervated preferentially the medial fibers showed a medial preference for new contacts; the neuron that normally innervated the lateral fibers showed a lateral preference for new contacts; the neuron that normally innervated everywhere regenerated equally well into both medial and lateral fibers. Therefore, these neurons are able to detect information regarding their position within the muscle mass and respond to it by preferential synapse formation. The effect of a positional gradient could not be detected when half of the target field was removed prior to regeneration. In this instance, the neuron that innervated the missing target area now regenerated to almost all the available fibers. It is suggested that the interplay of positional cues with other factors at different points in time could determine the final connectivity patterns formed by these cells.     

A model for budding in hydra: pattern formation in concentric rings

Current models of pattern formation in Hydra propose head-and foot-specific morphogens to control the development of the body ends and along the body length axis. In addition, these morphogens are proposed to control a cellular parameter (positional value, source density) which changes gradually along the axis. This gradient determines the tissue polarity and the regional capacity to form a head and a foot, respectively, in transplantation experiments. The current models are very successful in explaining regeneration and transplantation experiments. However, some results obtained render problems, in particular budding, the asexual way of reproduction is not understood. Here an alternative model is presented to overcome these problems. A primary system of interactions controls the positional values. At certain positional values secondary systems become active which initiate the local formation of e.g. mouth, tentacles, and basal disc. (i) A system of autocatalysis and lateral inhibition is suggested to exist as proposed by Gierer and Meinhardt (Kybernetik 12 (1972) 30). (ii) The activator is neither a head nor a foot activator but rather causes an increase of the positional value. (iii) On the other hand, a generation of the activator leads to its loss from cells and therewith to a (local) decrease of the positional value. (iv) An inhibitor is proposed to exist which antagonizes an increase of the positional value. External conditions like the gradient of positional values in the surroundings and interactions with other sites of morphogen production decide whether at a certain site of activator generation the positional value will increase (head formation), decrease (foot formation) or increase in the centre and decrease in the periphery thereby forming concentric rings (bud formation). Computer-simulation experiments show basic features of budding, regeneration and transplantation.     

The urodele limb regeneration blastema. Determination and organization of the morphogenetic field

The idea that the undifferentiated limb regeneration blastema of urodele amphibians is an undetermined and pluripotent structure is examined. A detailed review of the literature shows that this notion has no basis in fact. The data show that the morphogenetic potency of the blastema is restricted to its prospective significance and that this potency can be fully expressed when the blastema is transplanted either to a neutral location or to a regenerating organ of another type. Within this morphogenetic constraint, however, blastema cells have a histogenetic potency that is, at least in some cases, greater than their limb cell phenotype of origin. The morphogenetic responses of the regeneration field to discontinuities suggest that its autonomous determining relationships are based on the inheritance, from parent limb cells, of a graded set of mesodermal positional values specifying the pattern of the amputation plane, and a single epidermal external boundary value. The dividing mesenchymal cells of the blastema change positional value to erase any discontinuity between themselves and the epidermis, and the epidermis acts as a stop signal to inform the mesenchyme when the regenerate boundary has been reached. In vitro experiments suggest that changes in mesenchymal positional value in response to discontinuity can be interpreted in terms of gradients of cell-cell adhesivity, and they focus attention on the importance of molecular studies of blastema cell surfaces for our future understanding of regeneration and morphogenesis in general.     

Pattern formation during insect leg segmentation: Studies with a prepattern of a cell surface antigen

Summary A monoclonal antibody (MAb) that binds to a cell surface antigen selectively localized to epithelial cells undergoing morphogenesis was used to study the segmentation of the growing embryonic leg of the cockroachPeriplaneta americana. The MAb labels circumferential stripes of cells at locations where invagination will occur to form the leg segments. The formation of these stripes precedes any morphological change in the epithelial layer or in individual cells. The temporal and spatial distribution of the antigen indicates the existence of a prepattern for leg segmentation, examination of which can give information about pattern generating mechanisms. Although highly stereotyped, the sequence in which the stripes appear does not follow a simple pattern proceeding in one direction along the proximal-distal axis. It is proposed that each stripe is a boundary in a positional field. Stripe formation leads to the division of the leg into a repeating series of identical positional fields. Three different mechanisms for the formation of stripes of MAb labeled cells have been observed and the role of each in the evolution of the insect leg is discussed. Measurements of leg and leg segment lengths when the various stripes appear has demonstrated considerable variation, particularly at the early stages of segmentation. Rules or mechanisms generating pattern at early stages of development are not rigid. Variations arising are compensated for by later occurring events so that stereotyped structures are formed.     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Positional information and patterning revisited

The concept of positional information proposes that cells acquire positional values as in a coordinate system, which they interpret by developing in particular ways to give rise to spatial patterns. Some of the best evidence for positional information comes from regeneration experiments, and the patterning of the leg and antenna in Drosophila and the vertebrate limb. Central problems are how positional information is set up, how it is recorded, and then how it is interpreted by the cells. A number of models have been proposed for the setting up of positional gradients, and most are based on diffusion of a morphogen and its interactions with extracellular molecules. It is argued that diffusion may not be reliable mechanism. There are also mechanisms based on timing. There is no good evidence for the quantitative aspects of any of the gradients and details how they are set up. The way in which a signalling gradient regulates differential gene expression in a concentration-dependent manner also raises several mechanistic issues.     

Dermatoglyphic changes in human genetic disorders: preaxial defects of the upper limbs]

Dermatologlyphic prints of 9 patients with preaxial (radial) hand defects were compared with control ones. A correspondence is revealed between a decrease in thumb phalanx length and a respective decrease in ridge count, on the one hand, and between the 1st metacarpal hypo- or aplasia and a decrease in palmar ridge count between the metacarpo-phalangeal and thumb flexion creases, on the other hand. An interrelation is found between the anomalies in flexion crease and respective joint formation. It is suggested, that these disorders are due to genetically determined anomalies in morphogenetic gradients which control the distribution of positional information in upper limb morphogenetic field.     

Regeneration of Sclerenchyma in Wounded Dicotyledon Stems

When the sclerenchyma cylinder that surrounds the vascular cylinderin many dicotyledon stems is interrupted by cutting away oneside of an internode, its continuity becomes restored in somespecies by the differentiation of sclereids within the woundcallus. These sclereids, which may be scattered or arrangedin clumps or in a continuous sheet, lie in a zone within the‘cortical’ parenchyma between the regenerated vascularcylinder and the wound cork. The amount and especially the arrangementof regenerated sclerenchyma tends to reflect that of the originalprimary sclerenchyma cylinder in the unwounded stem, exceptthat longitudinal continuity is poorly developed and all fibresare replaced by sclereids. Syringa vulgaris L, lilac, regeneration, differentiation, fibres, sclereids, sclerenchyma, positional control     

Genetically Induced Abnormalities in Drosophila: Two or Three Patterns?

SYNOPSIS. The removal of a portion of a Drosophila imaginaldisc stimulates pattern regulation whereby the missing portionis regenerated or extra copies of the surviving portion (duplicationsand triplications) are produced. The results of recent experimentsin which temperature-sensitive cell-lethal mutations were usedto induce pattern regulation are considered here in an attemptto answer a major question: What is the nature of the pattern-formingsystem in the newly formed tissues? One current hypothesis isthat the observed pattern regulation phenotypes are the resultof interactions between portions of a single pattern. The evidencefrom studies in which Drosophila genetic investigative techniqueswere applied to developing leg duplications indicate that initiationand growth of the duplicate are similar or identical to normaldevelopment in terms of cell number, growth rate and patternof cell lineage (compartments). This suggests that portionsof the genetic mechanism underlying normal development may bereactivated and used to control abnormal development. Leg triplicationsconsist of one complete set of leg structures and two partialsets arranged in a mirror symmetrical cuticular process. Theseprocesses either become more or less complete distally (theydiverge or converge, respectively) by the addition or deletionof structures at the lines of symmetry. Whether a particularprocess diverges or converges is related to its circumferentiallocation. These leg duplication and triplication phenotypescan be explained as the result of cell death in situ followedby interaction between the surviving portions of the originalleg pattern following the rules of the polar coordinate modelof positional information.     

Simpler rules for epimorphic regeneration: The polar-coordinate model without polar coordinates

The polar-coordinate model of French, Bryant & Bryant (1976) describes epimorphic regeneration in insects and amphibians, and correctly predicts some surprising phenomena. Their model rests on two main rules. It is shown here that the first of these, the Rule of Intercalation, expresses a requirement that the pattern of positional values of the cells shall be continuous, and that the other, the Rule of the Complete Circle, is not an independent hypothesis, but simply a consequence of the continuity requirement. The formulation of the rules in terms of continuity is coordinate-free and applicable in any number of dimensions: polar coordinates do not have the fundamental significance attached to them by French, Bryant & Bryant (1976), nor are there any grounds to think that the system of positional values in an amphibian limb is two-dimensional. Some simple topology helps to clarify the concepts and prove these points. The phenomena codified by French et al. are to be expected in any epimorphically regenerative organ whose pattern of growth and pattern of positional values are defined by a maintained system of diffusible chemical signals.