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Abnormal regeneration in the planarianDugesia tigrina as a function of the length: Width ratio of the regenerating fragment
Abstract
Regeneration was examined in different regions of planaria (Dugesia tigrina) in order to determine the effect that the shape of a section exerts on regenerative success. Length:width ratios were used as an index of tissue proportion, and overall regenerative success was measured by percent of abnormal regenerates and number of abnormalities per abnormal regenerate. Frequency and number of abnormalities increased as section width exceeded section length. Frequency of specific abnormalities varied with changes in length:width ratios and followed a predictable pattern. As ratios decreased below 1.0, abnormalities typically consisted of characteristics regarded as being due to excess head formation. These included the presence of two heads, the “head hump” syndrome, and/or lack of pharynx. This excess head formation was due to the proportional shape of the section rather than to overall segment size, and the frequency of such abnormalities increased in proportion to decreases in length:width ratios. Abnormalities which were more typically seen at ratios over 1.0 consisted of the lack of head, pharynx, and/or polarity.
Regeneration was examined in different regions of planaria (Dugesia tigrina) in order to determine the effect that the shape of a section exerts on proportion regulation during regeneration. Length: width ratios were used as an index of section shape and experiments utilized sections at which this ratio was below 1.0 Proportion regulation was evaluated by determining relative area of major body regions for normal and abnormal regenerates. Proportional area of body regions anterior to the pharynx increased with proportional decreases in the length: width ratio for all regenerates, but this increase was greater for regenerates that originated from segments anterior to the pharynx. Changes in proportional area of one anterior body region were closely correlated to changes in porportional area of other anterior body regions. The exact nature of these correlations varied as a function of originating segment. A hierarchy also exists in proportional head tissue between normal and abnormal regenerates as total relative head area of twoheaded regenerates was around three times that of normal planarians while regenerates with one large head and without a pharynx had a proportional head size that was less than that of two-headed animals but more than twice that of normal planarians.
The time interval between cuts that are made to obtain a tissue fragment from a planarian was found to be important to the process of its regeneration. Short fragments made by two transverse cuts across the body were more likely to regenerate abnormally when the interval between the two cuts was 5 or 12 min than when it was 1.5 min. The longer intervals specifically altered the regression line in the correlation between the length:width ratio of fragments and frequency of abnormal regenerates. This effect occured regardless of which region of the body the fragment was taken from. The time interval also affected body proportioning in regenerates and to the greatest degree in fragments derived from the region located immediately behind the head. These results indicate that events occuring shortly after a cut is made in a planarian significantly affect structure patterning and proportioning of the regenerate.
Planarian regenerates with abnormal body proportioning were followed after structure formation in order to determine if body
proportion will ‘normalize’ over time. Results show proportioning will occur following structure formation since the pharynx
in regenerates with proportionally larger heads and prepharyngeal area moved anteriorly over time. This occurred regardless
of whether regenerates were provided with a normal feeding regime which allowed for an increase in body area or if they were
on a maintenance diet which did not permit growth. An examination of mitotic indices did not demonstrate significant differences
in the level of mitotic activity between pre- and postpharyngeal regions. It is concluded that the ‘normalizing’ of proportion
in these abnormal regenerates does occur, but the process by which it occurs is not solely explained through normal growth.
Studies on intercalary regeneration in several organisms have shown that a regenerate is formed when surfaces of different positional value along the proximo-distal axis are opposed. One of the main problems posed by this phenomenon is to know which piece contributes to the building of the regenerate. In the present work we have studied this problem in planarians using chimaeras made between pieces of different body levels, irradiated or not, of the sexual and asexual races ofDugesia(S)mediterranea that differ in a chromosomal marker.
The results found show very clearly that intercalary regenerates in planarians are formed by cells coming from both pieces (stumps), and that irradiated pieces keep the positional values and interact with non-irradiated pieces to restore the missing parts. This means that distal and proximal transformation do actually occur at the same time during intercalary regeneration in planarians. The implications of these results as regards to the origin of cells in the regenerate and to present models of intercalary regeneration are discussed.
The aim of the present experiments was the analysis of possible dorso-ventral regulation in planarians. The experiments employ "half-thickness pieces" obtained by a horizontal cut between the ventral and dorsal faces. When the dorsal face of an anterior part was superposed onto the ventral face of a posterior part (or vice versa) with its antero-posterior polarity reversed, both faces maintained their orgginal organization. When half-thickness pieces of the same face (dorsal or ventral) were fused by their transverse cut surfaces and cultured in vitro, intercalary regeneration ensued as it does in full-thickness pieces combined in the same manner. When a half-thickness piece was grafted to a distant site on the same face, the ensuing remodelling of tissues proceeded in the same manner as in similar experiments carried out earlier with full-thickness grafts. When full-thickness pieces representing different antero-posterior levels were joined so that the dorsal face of one fused with the ventral face of the other, intercalary regenerates were formed as if the pieces had been joined by their homologous faces-unless and terminal blastema appeared early on the suture. These experiments show that (1) there is no dorso-ventral self-regulating system: (2) the antero-posterior system resides in the periphery of the worm.
Regeneration was examined in fissioned and unfissioned planarians (Dugesia tigrina) in terms of regenerative success and body proportioning. Regenerative success was determined in terms of percent of abnormal regenerates and number of abnormalities per abnormal regenerate. Sections from unfissioned animals were less successful at regeneration than were those from fissioned animals, and body regions located immediately behind the pharynx represented those segments which formed fewer abnormal regenerates as a result of fissioning. Body proportioning was measured as a function of size of head and prepharygeal region to overall body length. A direct correlation was seen in the proportional size of these two parameters, which varied significantly among regenerates. Results suggest that body proportioning is not constant from one excised piece to another but is predictable and dependent on several variables including interval between fission and sectioning, region from which the segment was obtained, and/or original length of the segment.
The purpose of the present work was to obtain intercalary regeneration after joining two medio-lateral levels which are widely separated in the normal planarian. In all cases no intercalary regenerate was formed. Two parts of the same medio-lateral levels (medial or close to the lateral margin) belonging to different antero-posterior levels (prepharyngeal and postpharyngeal) were also joined. Intercalary regeneration ensued without exception. It was accomplished by the morphallactic remodelling of the prepharyngeal tissues, with a 90 rotation of their polarity. Together with previous observations these results suggest that there are two distinct self-regulating systems. The antero-posterior system (specifying antero-posterior levels) probably resides in a tissue in which the cells are continuously renewed, while the medio-lateral system (specifying medio-lateral levels) probably resides in a tissue in which the cells are able to de-differentiate.
In the planarianDugesia lugubris, when two originally widely separated body levels are joined together, intercalary regeneration is induced. The whole sequence of levels normally intervening between the two levels joined are reformed by one of the two associated pieces. Generally regeneration is accomplished by morphallactic remodelling. This process starts at the margin of the suture, which was originally nearer to the head, and progressively extends through the piece, which is entirely remodelled if it is too short. Thus, a head cut at the level of the eyes and joined to a tail is totally reshaped, forming a new head with a new pair of eyes and a new prepharyngeal zone in which the original eyes persist. When the head piece is too short, the pharynx is not produced by the regenerate, but secondarily through remodelling of the tail piece. Remodelling of the head piece is also observed when it is joined to a prepharyngeal piece. When a head piece is joined in reverse orientation to a tail piece, the remodelling, which is directed by the tail, leads to the reversal of polarity in the head tissues. When the head piece is entirely remodelled it forms an anterior extremity, a new head with new eyes and a prepharyngeal zone containing the original eyes. After joining the preocular level to a prepharyngeal level the intercalary regenerate is entirely built up by dedifferentiated cells (epimorphosis), which are produced by the prepharyngeal tissues (the margin which represents the more posterior level). The results do not support Child''s concept of dominance and are interpreted in the light of the concept of cell sociology.
The precision with which an almost uniform sheet of hydra cells develops into a complete animal was measured quantitatively. Pieces of tissue of varying dimensions were cut from the body column of an adult hydra and allowed to regenerate. The regenerated animals were assayed for number of heads (hypostomes plus tentacle rings), head attempts (body tentacles), and basal discs. To ascertain whether the head and body were reformed in normal proportions, the average number of epithelial cells in the heads and bodies was measured. Pieces of tissue, from to an adult in size, formed heads that were a constant fraction of the regenerate. Thus, over a 10-fold size range, a proportioning mechanism was operating to divide the tissue into head area and body area quite precisely, but appeared to reach limits at the extremes of the range. However, the regenerates were not all normal miniatures with one hypostome and one basal disc. As the width-length ratio of the cut piece was increased beyond the circumference-length ratio of the intact body column, the incidence of extra hypostomes in the “head” and body tentacles and extra basal discs in the “body” rose dramatically. A proportioning mechanism based on the Gierer-Meinhardt model for pattern formation is presented to explain the results.
Lateral fragments which contained no nerve cord were isolated from the postpharyngeal body section of Dugesia dorotocephala and fused with nerve cord grafts soon after isolation and at daily intervals through 8 days of regeneration. Fragments fused soon after isolation formed ‘headless’ regenerates but had normal body proportions. If the lateral cordless fragment was allowed to regenerate for 1 day or longer before fusion with the nerve cord fragment, the head always developed and the body proportions were normal. Therefore, head structures become determined in the lateral fragment within the first 24 h of regeneration; during this time these tissues can also respond to the head-inhibiting influence of the nerve cord. The competence to form particular structures of the postcerebral body regions must emerge after head-forming competence is lost, that is about 24h after isolation; however, it persists at least through the first 8 days of regeneration. Normal body proportions can be induced by nerve cord grafts throughout the first 8 days of regeneration.
Lateral fragments fused at any time after isolation with another fragment containing no nerve cord developed head structures but failed to differentiate tissues of the postcerebral regions. This confirms that the nerve cord is responsible for inhibition of head structures and induction of differentiation of body regions and normal body proportions.
Lateral cordless fragments isolated from the postpharyngeal section of Dugesia dorotocephala formed a large normal head at a 90° angle to the original antero‐posterior polarity; postcerebrally, only a hump of undifferentiated tissue developed. This “head‐hump” pattern, and also other types observed in previous studies of lateral fragments, were attributed to the absence of the nerve cord. In order to confirm the inductive role of the nerve cord and to eliminate the possibility that the “head‐hump syndrome” was due to the relative proportions of other tissues besides nerve, body fragments of two experimental groups were observed: (1) the five types of fragments which had no nerve cord but had varying proportions of other tissues present formed primarily “head‐hump” types of regenerates. (2) Almost all fragments which had varying amounts of nerve cord present but the same proportions of other tissues formed regenerates of normal body proportions. Therefore, the absence of the nerve cord does determine the “head‐hump syndrome”.
Isolated postpharyngeal half segments containing one nerve cord were allowed to regenerate for varying periods of time before the lateral cordless fragment was isolated. The number of “head‐hump” regenerates from lateral fragments isolated after a one‐day or longer contact with the nerve cord gradually decreased, and the number of regenerates with incomplete head development or which were more elongated postcerebrally increased. These results indicate that the nerve cord acts gradually to determine the differentiation of specific tissues rather than rapidly to determine the overall body plan.
By the use of a simple nutrient medium containing only amino acids, glucose, and inorganic salts, complete cephalic regeneration has been obtained from fragments of the planarian Dugesia dorotocephala as small as 0.08 mm3. It is calculated that this volume is equivalent to 1 x 104 cells. This number is very close to the minimal number of cells required for regeneration of Hydra and probably is a reflection of the minimal number of units or volume necessary to establish morphogenetic heterogeneities.
An inroad into an understanding of the complex molecular interactions on which development is based can be achieved by uncovering the minimum requirements that describe elementary steps and their linkage. Organizing regions and other signaling centers can be generated by reactions that involve local self-enhancement coupled to antagonistic reactions of longer range. More complex patterns result from a chaining of such reactions in which one pattern generates the prerequisites for the next. Patterning along the single axis of radial symmetric animals including the small freshwater polyp hydra can be explained in this way. The body pattern of such ancestral organisms evolved into the brain of higher organisms, while trunk and midline formation are later evolutionary additions. The equivalent of the hydra organizer is the blastopore, for instance, the marginal zone in amphibians. It organizes the anteroposterior axis. The Spemann organizer, located on this primary organizer, initiates and elongates the midline, which is responsible for the dorsoventral pattern. In contrast, midline formation in insects is achieved by an inhibitory signal from a dorsal organizer that restricts the midline to the ventral side. Thus, different modes of midline formation are proposed to be the points of no return in the separation of phyla. The conversion of the transient patterns of morphogenetic signaling into patterns of stable gene activation can be achieved by genes whose gene products have a positive feedback on the activity of their own gene. If several such autoregulatory genes mutually exclude each other, a cell has to make an unequivocal decision to take a particular pathway. Under the influence of a gradient, sharply confined regions with particular determinations can emerge. Borders between regions of different gene activities, and the areas of intersection of two such borders, become the new signaling centers that initiate secondary embryonic fields. As required for leg and wing formation, these new fields emerge in pairs at defined positions, with defined orientation and left-right handedness. Recent molecular-genetic results provide strong support for theoretically predicted interactions. By computer simulations it is shown that the regulatory properties of these models correspond closely to experimental observations (animated simulations are available at www.eb.tuebingen.mpg.de/meinhardt).
Reconstitution of lateral pieces of Planaria dorotocephala and Planaria maculata
- K. M. Beyer
- C. M. Child
Biology of the Turbellaria
- T. Lender
Proximal and distal transformation during intercalary regeneration in the planarian Dugesia (S) mediterranea. Evidence using a chromosomal marker
- E. Saló
- J. Baguà
Tumeurs spontanée chez la planaire Dugesia tigrina
- Stéphan F.
- Child
- Beyer
- Morgan