Prospective fates and regulative capacities of fragments of the female genital disc of Drosophila melanogaster.

A fate map of the female genital disc of Drosophila melanogaster was established by examining the derivatives of fragments transplanted into host larvae for metamorphosis. The fate map is presented as a two-dimensional projection, but for several reasons it is proposed that the anal plates originate from the dorsal epithelial layer whereas the genitalia are produced from the ventral layer. Fragments produced by cuts parallel to the axis of symmetry of the disc undergo regeneration during culture in adult hosts if the fragments comprise more than half of the disc, or duplication if they comprise less than half. Most of the fragments generated by bilaterally symmetrical cuts across the line of symmetry of the disc undergo neither regeneration nor duplication during culture, but with some such fragments there is a low frequency of regeneration. It is argued that the usual lack of regeneration in these fragments results from wound healing which confronts identical positions from right and left sides, giving no growth stimulation. The fragments which regenerate might do so as a result of healing between dorsal and ventral surfaces, providing the discontinuity in positional information which is thought to be involved in growth stimulation.     

Wound healing and regenerative response of fragments of the Drosophila wing imaginal disc cultured in vitro

Fragments of imaginal discs of the fruitfly Drosophila undergo growth and pattern regulation when cultured in vivo in adult female hosts for several days prior to metamorphosis in host larvae. Pattern regulation results in either regeneration of excised pattern elements or duplication of elements whose fate map positions lay within the fragment. Initial wound healing along the cut edge of a fragment is thought to be a crucial first step in the process of pattern regulation. We have examined the capacity for wound healing and pattern regulation of fragments (distal halves) of the wing disc cultured in vitro, using the culture system recently reported to support extensive growth and transdetermination of slightly wounded whole imaginal discs in vitro. Our results suggest that disc fragments and whole discs apparently respond differently in the culture system. With disc fragments, wound healing did not occur in vitro. When fragments were first cultured overnight in adult female hosts to allow initial wound healing prior to explantation in vitro, then some volume increase and regeneration of excised portions occurred during 2–3 weeks of culture in vitro. The extent of apparent growth was much less than that reported for whole discs, and the frequency of regeneration in vitro (19%), while highly significant relative to controls not cultured in vitro (0%), was much less than that observed for fragments cultured in vivo (84%). Furthermore the extent of regeneration which occurred in vitro was considerably smaller than that which occurs during regeneration in vivo.     

Positional information in imaginal discs transformed by homoeotic mutations

Summary Mutations of the bithorax complex result in segmental transformations in the thorax and abdomen ofDrosophila. The haltere discs from larvae homozygous forbx ³ orpbx are transformed so that the discs contain cells that will produce wing cuticle as well as cells that produce haltere cuticle. The pattern regulation behavior of these discs has been examined. The fate maps of the two discs were established, and then the regulative behavior of a number of fragments from both types of mutant discs was established by culturing the fragments in vivo prior to metamorphosis. The most important conclusion from this work is that the cells producing, haltere cuticle and wing cuticle within the same disc share the same positional information and that they communicate during pattern regulation.     

Sexual homologies and intercalation between parts of the male and female genital discs of Drosophila melanogaster.

To compare the homologies in distribution of positional values within the male and female genital discs, bilaterally symmetrical (horizontally cut) fragments were isolated and combined by mixing them together with tungsten needles. The combinations were then cultured in adult hosts before transfer to larvae for metamorphosis. Fragments that showed no regeneration when cultured intact or when mixed with identical fragments could be induced to regenerate by mixing them with different parts of the same disc. Fragments that showed occasional regeneration in controls were induced to regenerate at higher frequencies in such mixtures. Regeneration could also be induced or increased in frequency by mixing with certain fragments of the genital disc of the opposite sex. It is argued that the extra cases of regeneration in the mixed combinations are due to intercalation between the two components in the combination, and a detailed model is presented to account for the results. It is suggested that the male and female genital discs begin development with identical arrays of positional information, but that different parts of the field grow in the two sexes. The part which does not grow might be retained in the disc to provide a continuous field of positional information, although it would not produce any adult derivatives.     

Is regeneration inDrosophila the result of epimorphic regulation?

Summary It has been known for many years that when a wing disc ofDrosophila is bisected, and the fragments cultured in adult females, regulation occurs and either a complete disc is regenerated or the fragment is duplicated. We have investigated how this regeneration process occurs. To establish which cells contribute to the regenerate, and thus determine if regeneration is the result of epimorphic regulation, fragments of discs, after culture in an adult for one to five days, were exposed to³H-thymidine to label replicating cells. Imaginal discs, both whole and as regenerating fragments, undergo some DNA replication which is distributed throughout the disc, but cut discs frequently show clusters of labelled cells around the wound, indicating that regeneration is probably epimorphic.     

Regeneration in youngBunodactis verrucosa Pennant (actinaria,anthozoa)

Summary YoungBunodactis verrucosa Pennant at the 12 tentacle stage are employed to test the applicability of the polar coordinate model to coelenterate regeneration. The animals are cut along every radius into fragments of 3 to 9 segments. Most fragments are patent 3–4 weeks later, but small fragments have a higher mortality rate than large fragments. Some fragments do not regenerate and occasionally tentacles fuse, thereby reducing the number of segments. Small fragments tend to regenerate more tentacies than large fragments, but large fragments may regenerate great numbers of supernumerary tentacles. Twenty-two percent of the fragments restore the missing number of tentacles, while 76% of all fragments produce an even number of tentacles.Fragments restoring the correct numbers of tentacles show a marked tendency to form the correct tentacles (regulative regeneration). Fragments regenerating two less than the number of tentacles already present show a marked tendency to reproduce tentacles of the types already present (miror image formation). Other fragments produce missing segments (forward regeneration), or those already present (reverse regeneration) at lower frequencies.No fragments beginning or ending with the number 1 directive tentacle fail to regenerate entirely, while first cycle segments maximally remote from segment 1 are associated with the absence of regeneration. No fragments beginning or ending with the number 4 directive tentacle fail to undergo forward regeneration, regulate or produce a mirror image when the appropriate number of segments are regenerated. In contrast, segment 4 is associated with a low frequency of reverse regeneration, and second cycle segments cut away from immediate contact with segment 4 show an increase in the frequency of reverse regeneration. Controls through morphogenic substances rather than polar coordinates seem to explain these results. Such substances would control the number and direction of tentacle regeneration.This work was performed while the author was on sabbatical leave from the University of Pittsburgh at the Stazione Zoologica di Napoli. The author gratefully acknowledges the assistance of Mr. Ciro Gargiulo and of Ms. Gisella Princivalli. This work was supported by a travel grant from the United States Italy Cooperative Science Program of the National Science Foundation. The paper is dedicated to Dr. Alberto Monroy whose generosity made it possible     

Leg regeneration in insects. An experimental analysis in Drosophila and a new interpretation.

Fragments from prospective distal regions of Drosophila male foreleg imaginal discs failed to undergo proximal intercalary regeneration across leg segment borders when mechanically intermixed and cultured for 8 days with various fragments from prospective proximal disc regions. The failure of the distal cells to regenerate proximal leg segments was not due to a general restriction in their developmental potentials: Distal fragments, when deprived of their distal-most tips, regenerated in the distal direction at a high frequency. It is concluded that there exist in Drosophila leg discs the same restrictions with respect to regeneration along the proximodistal leg axis as had been previously observed in legs of several hemimetabolous insect species: Intersegmental discontinuities between grafted tissue pieces are not eliminated by intercalation. Based on the available evidence in hemimetabolous insects and in Drosophila, a new interpretation of the different aspects of regeneration in insect legs is offered. It is proposed that the two categories of regulative fields observed in insect legs, the leg segment fields and the whole leg field, represent the units of regulation for two fundamentally different regulative pathways that a cell at a wound edge can follow, the intercalative pathway and the terminal pathway, respectively. It is suggested that the criterion used by cells at healing wounds to choose between the two pathways is the difference in circumferential positional information between juxtaposed cells. The intercalative regulative pathway is switched on when cells with disparities in their axial positional information, or cells with less than maximal disparities in their circumferential information, contact one another. The terminal regulative pathway is triggered whenever cells with maximal circumferential disparities come into contact.     

Regulative Properties of Wing Discs from the Vestigial Mutant of Drosophila melanogaster

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult.
Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.     

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.     

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.     

Differentiation Capacities of the Foreleg Imaginal Disc of Sarcophaga ruficornis Fabr (Diptera, Sarcophagidae): I. Fate Map

When an imaginal disc of a mature larva is implanted into a host larva of the same age, it undergoes metamorphosis with the host. On the basis of the results obtained from the transplanted whole disc and disc fragments, a fate map of the foreleg disc of Sarcophaga ruficornis has been constructed. The fate map of S. ruficornis presented by us is basically similar to that of Drosophila but anterior and posterior rows of bristles of femur, apical, preapical and one long bristle of tibia have been precisely localized.     

Regeneration and duplication following operations in situ on the imaginal discs of Drosophila melanogaster

A technique is described which allows defects to be made in situ in the imaginal discs of immature Drosophila larvae. Bisection of the second leg disc across the upper-lower axis results in regeneration of the remainder of the disc from the upper portion, and mirror-image duplication of the anlagen in the lower half (Figs. 2–5, Table 1). The upper half, retaining its connection to the larval epidermis, is able to evert; the lower half metamorphoses as an uneverted implant. Partial bisection of the disc often results in the production of branched legs in which one branch is complete but the other is a double half (Figs. 7–9). These cases can be interpreted as resulting from regeneration from one cut surface and duplication from the other. Pattern triplications have been obtained by partial bisections of the wing disc (Fig. 10), and these can be interpreted in a similar manner. It is suggested that regeneration and duplication are identical phenomena, resulting from the properties of of the anlagen at the cut edge. Cases of regeneration and duplication in other insect and vertebrate systems are discussed, and interpreted on the basis of gradients of developmental capacity (Fig. 11).     

Wound healing and regeneration in the imaginal wing disc ofDrosophila

Summary When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In 06 fragments (Bryant 1975a) wound healing has occurred after 1–2 days of culture and the regeneration of missing structures after 2–4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days.When disc fragments are cultured in female abdomens and then exposed to³H-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication.     

Wound healing in the imaginal discs of Drosophila. I. Scanning electron microscopy of normal and healing wing discs.

Scanning electron microscopy was used to investigate the morphology of intact imaginal wing discs of third-instar larvae of Drosophila melanogaster. The disc stalk, nerve and tracheal entries and the surface ultrastructure of the columnar cells, the peripodial membrane cells, and the adepithelial cells are described. The behavior of various fragments of the wing disc during culture in vivo was also studied. After injuring a wing disc by cuts with a tungsten needle, during the first day of culture the epithelium curls and the wound surface contracts. Subsequent closure of the wound in $\text{3}\text{4}$ and $\text{1}\text{4}$ sectors, in fragments generated by straight cuts and in central squares, leads to the confrontations of cells from formerly separate positions, as was proposed in connection with the polar coordinate model of French, Bryant, and Bryant [(1976). Pattern regulation in epimorphic fields. Science193, 969–981]. Wound healing comprises three steps: (1) Cell debris is removed; (2) occasional cell processes span the wound; (3) all cells at the wound edge contact cells on the opposite side. After 2–3 days, a continuous epithelium is re-established. The tissue distortion may lead to transient contacts of the columnar epithelium with the peripodial membrane and with itself. The latter can explain the occasional duplications of structures which, according to the fate map, arise from near the wound edge, and which have been previously reported from cultured imaginal disc fragments. The tissue movements appear to be due to the contractile properties of individual cells.     

Participation of lethally irradiated imaginal disc tissue in pattern regulation in Drosophila

When fragments of tissue derived from opposite ends of an imaginal disc are mixed prior to culture, intercalary regeneration occurs so that structures are produced which neither of the fragments would have made when cultured alone. We report here that, if a fragment is irradiated so heavily with X rays that its cells do not survive culture and metamorphosis, it nevertheless can stimulate regeneration by a fragment with which it is mixed.     

High cell‐autonomy of the anterior endomesoderm viewed in blastomere fate shift during regulative development in the isolated right halves of four‐cell stage Xenopus embryos

The isolated right half (RH) or left half (LH) of Xenopus embryos can undergo regulation so as to form well‐proportioned larvae. To assess how the combined actions of maternal determinants and cell–cell interactions contribute to form the well‐proportioned larvae, we quantitatively compared four‐cell stage blastomere fate between normal larvae and regulated larvae from RH embryos. In normal larvae, the clones of the right dorsal blastomere (RD) and right ventral blastomere (RV) were located unilaterally. In contrast, in regulated larvae: (i) the RD clone exclusively occupied the anterior endomesoderm (AE) derivatives, coinciding no RV progeny in those derivatives of normal larvae. The clone bilaterally populated tissues along the dorsal midline, which characteristically included the medial regions of both somites adjoining the notochord, with higher percentages on the right and anterior sides. (ii) The RV clone extensively compensated for the missing left side at the expense of its right side contribution, and bilaterally occupied the ventroposterior and also dorsal regions excluding the AE derivatives. This clone considerably populated, with altered orientations, the derivatives of the left half gastrocoel roof plate (GRP), the left half GRP being essential for laterality determination. These results show that the high cell‐autonomy in the AE constitutes a mechanism common to both normal and regulative development. In regulated larvae, cell–cell interactions shifted the midlines on the dorsal side slightly and the ventral side to a greater extent. The cell lineage difference in the left half GRP could result in a different utilization of maternal determinants in that area.     

Morphological and somatic clonal analyses of pattern triplications

The growth of pattern triplications induced by a 48-hr 29 degrees C treatment given to larvae homo- or hemizygous for a ts cell-lethal mutation was examined to determine which structures result from new, regulative growth and which are produced by the original imaginal disc cells. Pattern triplications contain one complete leg pattern (orthodrome) and two partial patterns (antidrome and paradrome). The results of two morphological analyses and one somatic clonal analysis suggest that in triplications in which the antidrome and paradrome become more complete distally (diverge) the paradrome is formed by a portion of the original leg pattern, and the antidrome and orthodrome are formed by extra, regulative growth. A different result is suggested for triplications in which the antidrome and paradrome become less complete distally (converge). In these, the orthodrome appears to be formed by the original leg pattern and the antidrome and paradrome by extra growth. These results agree with predictions based on the polar coordinate model of positional information.     

Genetic Variation in Potato Cv. Record: Evidence from In Vitro 'Regeneration Ability'

Regeneration ability in vitro was studied in 170 individualtubers putatively derived from several or many parent plantsof the potato cv. Record. Of these, 120 were sprouted and thesprouts used to establish in vitro shoot cultures for leaf discproduction. The other 50 were grown in a glasshouse for theproduction of leaf discs. The reliable regeneration of somaclonesfrom leaf disc calluses was successful from only 11 parentaltubers. In ten of these, somaclones were derived from in vitroshoot cultures, and from a glasshouse-grown plant in the other.Four parental tubers gave the majority of somaclones, and one,R149, produced 85% of all somaclones at 15 months from initiationof leaf disc cultures. This differential regeneration abilitymay be due to genetic differences between tubers in this potatocultivar as it was found to be maintained in subsequent tubergenerations. The results are discussed in terms of seed potatoproduction and in vitro genetic conservation of vegetativelypropagated species. Potato, Solanum tuberosum cv. Record, regeneration ability, leaf disc culture, somaclonal variation     

Cell proliferation changes during pattern regulation in imaginal leg discs of Drosophila melanogaster

Upon fragmentation of a leg imaginal disc, cells near parts of the wounded surface are reprogrammed and form a blastema. This occurs without a change in fate and without the direct contact of the two wounded surfaces (G. H. Karpen and G. Schubiger, Nature (London) 294, 744-747, 1981). Two phases of the cell cycle have now been analyzed for several areas of disc fragments prior to and during wound healing. A mitotic index was used to compare the location of cell division, and autoradiography was used to reveal patterns of DNA synthesis. In contrast to the uniform division pattern in noncultured fragments, more dividing cells were observed near the two wound surfaces after 1 day of in vivo culture. During the second day, wound healing began and mitotic activity increased dramatically near both wound areas, and decreased in distant areas. Three and a half days of culture led to more complete wound closure and only cells on one site continued to show the highest frequency of labeled cells. It is concluded that changes in patterns of DNA synthesis and an increase in cell division begin prior to wound closure. This proliferation is consistent with the morphological changes and regulative behavior observed. In addition, the role of compartmental identity during regulation was tested. After wound closure began an increase in mitotic activity near wounds in the anterior compartment was observed whereas such an increase in division level was not seen in posterior cells near a wound.