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Master's Theses Theses and Dissertations

1974

Regeneration in Zebrafish, Brachydanio rerio

Gabriel J. Ekandem Loyola University Chicago

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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1974 Gabriel J. Ekandem REGENERATION IN ZEBRAFISH,

BRACHYDANIO RERIO

BY

GABRIEL J. EKANDEM

A THESIS SUBMITTED TO THE FACULTY OF THE

GRADUATE SCHOOL OF LOYOLA UNIVERSITY

IN PARTIAL FULFILLMENT OF THE RE~UIREMENTS

FOR THE DEGREE OF MASTER OF SCIENCE

JUNE, 1974

-..... · COMMITTEE IN CHARGE OF CANDIDACY

Dr. Harold W. Manner (Chairman}

' Dr. Amrik Dhaliwal

Dr. A. Rotermund

' ACKNOWLEDGEMENT

I would like to take this opportunity to thank Dr. Harold W.

Manner for his constant help and encouragement.

I also wish to thank Dr. A. Dhaliwal and Dr. A. Rotermund for their invaluable suggestions.

ii TABLE OF CONTENTS

Page

ABSTRACT •••• iii

INTRODUCTION •• . . . . 1

REVIEW OF THE LITERATURE 3

MATERIALS AND METHOD...... 71 EXPERIMENTAL RESULTS •...... 74

DISCUSSION . • 76

CONCLUSION ...... 80

LITERATURE CITED . . . ' . 81

APPROVAL SHEET .•• 119 ABSTRACT

The regeneration of the posterior region of Zebrafish,

Brachydanio rerio has been studied. The posterior region or tail, as used in this paper, means any point posterior to the cloaca.

The tail of Zebrafish demonstrates a notable capacity for self-replacement during the first month of its life. The initial direction of regenerative growth depends upon the angle of the amputation plane. If the cut is at an angle, rather than transverse, the axis of the regenerate is at right angles to the amputation plane.

Growth curve showed a lag period the first day of amputation, a· period of rapid growth lasting four days, and then a time of decelerating growth. Regeneration was completed on the 30th day following amputation.

iii INTRODUCTION

Two centuries ago, an Italian biologist, Lazzaro Spallanzani

published his famous "Prodromo di un Opera Sopra la Riproduzioni

, .... Anirriali~ 11 To this day this remarkable book stands as the foundation

upon which current regeneration research is based. He postulated that

fish do have some power of regeneration. Ten years later Broussonet

removed part of a fin and demonstrated that it did regenerate the

distally removed portion, thereby lending support to Spallanzani' s

postulation. The same experiment was repeated in the following year

by Philippeaux. He obtained the same result. These works opened the

field of fish regeneration to all developmental biologists.

CONANT (1970) working with the African lungfish Protopterus

showed that fish can regenerate not only tail fins but muscles, blood

vessels, vertebral column, connective tissue and pigment cells.

Several authors have studied regeneration in fish: fin regenera­

tion in teleosts, (Morgan, 1900); further experiments on the regeneration

of the tail fins in fishes, {Morgan, 1902); fin regeneration in lungfish

(Coats, 1937); the role of the central cartilaginous rod in the regenera­

tion of the catfish barbel, {Goss, 1954); taste barbel regeneration in

catfish, (Goss, 1956); the mechanisms of joint and bone regeneration in

the skeleton rays of fish fins, (Haas, 1962). Other workers in the field

1 2 of regeneration are: Tassava and Goss, (1966) analysed regeneration rate and amputation level in fish fins. Durand. (1960) analysed endocrine activity in fish regeneration. Kamrin and Singer, (1955) worked on the influence of the nerve on regeneration and maintenance of barbel in the catfish. Goodrich and Green. (1959), analysed the color pattern during regeneration in Brachydanio albolineatus.

This thesis has demonstrated that Zebrafish, Brachydanio rerio, when 10 days old, can regenerate not only fins but muscles. blood vessels. notochord and connective tissue. REVIEW OF LITERATURE

Many definitions of the phenomenon of regeneration are available.

However, there is always something common, constant and basic, and

this should be reflected in the main definition of regeneration. As long

ago as 1901 Morgan suggested that "Regeneration is the process by

which organisms replace structures or organs which have been lost by

accident or mutilation." More recently Goss (1967) suggested that

11Regeneration is rebirth or restoration of a lost or damaged part"

(large segments of the body, organs, parts of organs. tissues, cells,

parts of cells of the body, and in the present study entire posterior portion of fish's body).

Neither of these definitions are quite adequate because they do not cover two phenomena which belong undoubetedly to the category of regeneration phenomena. In some invertebrates, such as the fresh­ water polyp Hydra (Child. 1941). the planarian flatworm, Dugesia

(Bronstedt, 1955); Child, 1941; Morgan. 1901; Lund, 1947; Barth. 1955;

Wolff, 1962; Lender, 1962). and the starfish, (Berril, 1961; Hamburger,

1965), a small fragment of the body can restore a complete. whole organism rather than merely an organ. Furthermore, many animals can restore structures lost as part of the normal metabolic processes of life. The periodic molting of feathers in birds (Windle, 1955), the

3 4

shedding of fur in mammals (Thornton, 1959; Dunphy and Edwards,

1958; the replacement of the exoskeleton in arthropods (Needham,

1965; Bodenstein, 1955; Krishnakumaren and Scheiderman, 1964;

Winglesworth, 1964); and the renewing of the epidermal scales in

reptiles, (Woodland, 1920; Barber, 1944; Simpson, 1965); are examples

of physiological regeneration. The uppermost cornified skin layers in

mammals including man are constantly worn off and replaced by active,

proliferating cells from germinal layers of the skin. The same holds

for the cmtinuous replacement of hair {Woodland, 1920). nails (Barber,

1944) and claws. (Simpson, 1965); The teeth are replaced only once

in mammals, including man, but there is a continuous succession of

teeth in lower forms, such as the dogfish and shark (Goss, 1967; The

antlers of deer are shed at regular intervals and then regenerated

(Goss, 1963. The periodic changes in the genital tracts of female

mammals during menstruation and oestrus also should be included here.

All these instances in which replacements are part of the normal life

functions are called physiological or repetitive regeneration which fo~lows

injury. This paper deals primarily with the latter type.

Capacity for Regeneration

There are wide differences in the regenerative capacity of dif­ ferent animals. The one extreme is represented by those invertebrates

in which a part of the body can restore a whole organism. In higher

organisms, e. g., the Salamanders, (Singer, 1952; Zika and Singer, 1965; 5

Humphrey, 1966) and many crustaceans and insects, (Durand, 1960) only organs, such as limbs, can regenerate. Mammals cannot restore entire organs but they can repair damage to tissues, such as bone fractures, skin and muscle injuries and peripheral nerve loss (Singer,

1952). These phenomenon of tissue repair are, of course, included

. under the general heading regeneration.

Reparative regeneration is restoration for loss caused by trauma.

Trauma damage may be of different kinds: mechanical, chemical, thermal, radiational; it may be caused by the invasion of pathogens. The nature of the damage is of great importance for the subsequent process, but this circumstance does not alter the basic point that L'1. all such cases of repair, posttraumatic, nonphysiological regeneration takes place

(Goss, 1967).

Self-Amputation.

A number of animals, when attacked and caught by a leg or tail, save their lives by casting off this appendage. The organ which is sacrificed can usually be restored by regeneration. This act of self­ amputation is called autotomy (Needham, 1952). Instances of autotomy are found in a number of different animal groups, such as coelenterates

(Tardent, 1963; Steinberg, 1955; Burnett, 1962; Haynes, 1963); mollusks, echinoderms, annelids, arthropods and others (Hay, 1966; Herlant­

Meewis, 1964). This means of escape however is used by only a few representatives of these phyla. 6

The only case of autotomy in vertebrates, and perhaps the best known of all, is that of the autotomy of the tail in lizards (Moffat and

Bellians. 1964). The facility with which the tail breaks off is due to a special structural adaptation. When grasped, the tail separates along a breaking plane present at the base of the tail. Several vertebrae are split across the middle. The halves are held together by cartilage which ruptures readily. The regeneration, which usually begins at the breaking plane, produces a tail that is atypical. It contains no vertebrae but merely a cartilaginous skeletal axis. The muscle and nerve distribu­ tion is likewise atypical. Nevertheless, such a regenerated tail can regenerate a second time (Kamrin and Singer, 1955; Simpson, 1965).

Many crabs. insects and spiders have a similar device to facilitate autotomy, namely a preformed breaking plane at the base of legs or antennae. The chitinous exoskeleton is soft and thin at certain levels, and the muscle arrangement expedites self-amputation. It is not always the same segment of the leg at which such a breaking plane is prepared, but the mechanism is apparently similar in all species

(Waddington, 1956). In the walking stick, or stick insect, all three pairs of legs are adapted for autotomy. Quite frequently, special care is taken to avoid an excessive loss of blood. The skin contracts over the wound and closes it off (Balinsky, 1965). Some sea anemones release a tentacle when it is strongly stimulated; (Anderson, 1965) starfishes cast off an arm voluntarily, (Anderson, 1965) and some annelids can autotomize 7

the hindmost body segments and regenerate them subsequently, (Berril,

1961, Hamburger, 1965).

One of the most startling instances of autotomy is the self­

evisceration in sea cucumbers. which, when strongly stimulated, cast

off their anterior ends, including tentacles, mouth parts and the water­

vascular system. At the same time they discard, through the anus, the

intestine and attached structures, such as gonads (Berril, 1961;

Waddington, 1956). The discarding of the inner organs is accomplished

by strong muscle contraction. The nearly empty hull composed of skin

and muscles is capable of regenerating the autotomized organs. Thione

can accomplish this feat within a month (Berril, 1961).

There is only one step from spontaneous autotomy to spontaneous fission for the purpose of asexual reproduction. Starfishes and sea

cucumbers break apart at more or less regular time intervals and the fragments regenerate a whole individual (Anderson, 1965; Hyman, 1955). -

Likewise, in certain annelids and flatworms posterior parts of the body are constricted off and the fragments regenerate into new individuals.

This performance has been established as a regular mode of reproduc­ tion. The close affinity of regeneration and reproduction in lower animals is thus again affirmed.

Atypical Regeneration.

A regenerate is ordinarily a replica of the original structure. but a number of cases are known in which regeneration is atypical in 8 that either more or less than was lost is regenerated. Occasionally, the regenerate represents an organ entirely different from the one it replaces. All forms of atypical regeneration are called heteromor­ phosis, (Morgan, 1901; Child, 1965).

Incomplete Regeneration.

This type of heteromorphosis is common both in naturally occurring and in experimentally induced regeneration. Regenerated tails of lizards invariably show certain structural deficiencies, such as the absence of normal vertebrae. Regenerated salamander limbs frequently have a reduced number of digits or even greater deficiencies.

Those of arthropods may have a reduced number of segments. Several species of annelids can regenerate only a limited number of head segments.

If more are amputated, the total segment number of the worm after complete regeneration will be subnormal. Planarians occasionally regenerate a head with two fused eyes, or only one eye, or eyeless heads, instead of the normal head with two separate eyes (Thornton, 1959).

Such atypical head regenerates can be produced experimentally by exposing the regenerating animals to any one of a number of chemical agents which are known to impair developmental processes in a general, nonspecific way.

Superregeneration.

Of greatest interest among the super-regenerations are those in 9

which a single organ is replaced by a duplicated or multiple formation.

Instances of such monstrosities have been observed (Morgan. 1900).

In hydranths of Hydra and of its marine relatives, in heads and tails

of planarians and annelids, in tails of lizards and limbs or digits of

amphibians and particularly frequently in appendages of arthropods

(Child, 1941). Double claws of crabs and lobsters, and double legs

and antennae of beetles and other insects have been described (Morgan,

1901). Starfishes with bifurcated arms and sea cucumbers with dupli­

cated body parts also have been observed (Needham, 1965). Triplicate

appendages are commonest in arthropods and occasionally are found in

other forms. (Child, 1941). They originate probably in most instances

in the following way: a leg, claw or antenna ruptures at a joint without

breaking off completely (Bodenstein, 1955; Penzlin, 1964). As a result,

two wound surfaces are exposed, each of which begins to regenerate the

distal parts. The two new formations, together with the persisting

original structure, form the triple monstrosity. It was found that such

triplicate structures follow a definite rule of symmetry relations (Bateson's

rule): two adjacent components, namely, the middle one and one of the

marginal components, are mirror images of each other, whereas the two marginal parts have the same symmetry pattern (Barth, 1955).

It is often difficult or impossible to decide whether double or triple monstrosities found in nature have been brought about by regenera­ tion or whether the duplication occurred in early embryonic development. 10

Therefore, experimental methods were devised that make it possible

to study the origin of duplications by regeneration under controlled

conditions. An expedient way of accomplishing this is to create a

wound surface which will produce two separate blastemas instead of

one {Thornton, 1959).

If the tail of a tadpole is amputated by two oblique cuts, in

the form of an arrowhead {instead of by one transverse cut), then two

blastemas and tails will grow out, each with its main axis perpendicular

to the oblique, cut surface (Barfurth 's rule). Multiple digits in sala­

mander legs were produced in the same fashion, by making a lonitudinal

fission in the median plane through the anterior part of the animal, and

a subsequent amputation of the two halves of the two halves of the head.

Each separate anterior body half then regenerates the missing lateral

parts and, in addition, a whole head at each anterior surface. By

repeating this procedure, animals with multiple heads {up to ten) have been obtained. In the same way, planarians with double tails can be produced experimentally (Hamburger, 1965).

Homeosis.

This category of atypical regenerations includes all those instances in which the regenerate represents a structure different in type from the original {Morgan, 1901; Needham, 1965). Romeo sis occurs mainly in arthropods. It is a characteristic of this phylum that most of the numerous body segments bear appendages which are of different types in different 11 body regions (e. g., antennae, mandibles, claws, thoracic legs, abdominal appendages). In homeosis, the appendage of one type substitutes for another type. A few examples may serve as illustra­ tions: in crustaceans (crayfish, lobster, crab), a thoracic leg with claws was regenerated in place of a maxilliped; in other cases an abdominal leg was found in place of a thoracic leg, (Needham, 1965; Wigglesworth,

1964). A leg may be regenerated in place of an antenna or of a mandible, and vice versa. An anterior wing of a butterfly or moth may replace a posterior wing. Of particular interest is the regeneration of an antenna in place of a stalked eye, which was observed in marine decapod crustaceans of the genus Palinurus and Palaemon (Morgan, 1901).

Regeneration, as noted above, can occur by reorganization of the old piece (morphallaxis) (Child, 1941), or by outgrowth of new tissue at the cut surface (epimorphosis) (Thornton, 1960; Morgan, 1901). Only the later mode of regeneration is discussed in the following. In limb and tail regeneration of salamanders, in head and tail regeneration of plannrians and in many similar instances, a regeneration bud or blastema is formed at the amputation surface. The blastema is at first a slight elevation which grows to a cone-shaped structure and then begins to differentiate into tissues or organs. The blastema is an accumulation of cells of embryonic type, whose origin has been the topic of numerous investigations. The material that builds up the regenerate may be derived from three different sources. In several invertebrate phyla, 12

including coelenterates, annelids and flatworms, a special type of

undifferentiated cells, the so-called reserve cells or neoblasts, are

stored in different parts of the body. When the need for regeneration

arises they are mobilized and migrate to the site of injury. Tissue

outgrowth is a second source. The differentiated tissues at the cut

surface (for instance, skin or muscle) may simply grow out and each

form tissue of its own kind. Finally, the blastema cells may be

derived from formerly differentiated tissues of the amputation stump

which have dedifferentiated and returned to an embryonic-type condi­ tion. It is conceivable that when the blastema proceeds to develop,

such cells may differentiate into a type of tissue different from the one from which they were derived. For example, in salamander limb regeneration, a former muscle cell may become a bone cell, or vice versa. This transformation is referred to as metaplasea.

Tissue Outgrowth.

This mode of regeneration is characteristic of tissue repair such as bone regeneration following a fracture, restitution following a fracture, restitution of a muscle injury or liver regeneration. Peri­ pheral nerves regenerate in this fashion, by outgrowth from the cut end. In amphibian limb regeneration, apparently only nerves and skin regenerate in this way. The skeletal elements of the regenerate do not originate from old skeletal tissue at the cut surface. This was ... 13 demonstrated by an experiment in which the upper arm bone {humerus) was carefully removed and the limb then amputated across the upper arm.

The regenerated forearm and digits contained all typical skel­ etal elements, although no bone tissue was present at the amputation surface (Singer, 1952).

Reserve Cells (Neoblasts).

A glance at some of the extensive regenerations in lower animals shows that in these cases tissue outgrowth cannot be the primary source of the new formations. For example, a narrow piece from the middle of a planarian can regenerate a new individual, with such organs as brain, eyes, pharynx and reproductive organs, no trace of which was present in the original piece. In flatworms, annelids, coelenterates and tunicates the reserve cells or neoblasts are the major source of the building material for new organs. These cells have been set aside in the embryo for this purpose, and have never undergone any special­ ized differentiation. In the common coelenterate Hydra, which is famous for its regenerative capacity, a type of connective tissue or mesenchyme cell, the so-called interstitial cell, is found scattered throughout the body: these cells migrate to a cut surface and form the chief or even exclusive source of the blastema. In some annelids the neoblasts can be identified as particularly large cells, stored in differ­ ent parts of the body. However, it seems that tissue outgrowth and 14

metaplasia also contribute to regeneration in annelids, and there is a

· "· great deal of variation in details in this group (Gross, 1964; O'Steen,

1958; Thornton, 1960).

Different species of flatworms vary greatly in their capacity for

regeneration, and a correlation seems to exist between the degree of

Tegenerative power and the quantity of reserve cells present in the body.

The role of these reserve cells in planarian regeneration was firmly

established by the use of a modern tool, irradiation (Burnett, 1962;

Tardent, 1963; Wolff, 1962). It was found that reserve cells are more

sensitive than ordinary body cells to X-rays and other radiation. If

the whole animal is exposed to a mild dosage, the neoblasts can be des­

troyed without destroying the animal, which survives for at least sever·al

weeks. These individuals have no capacity for regeneration. A Modifica­

tion of this experiment gave evidence that reserve cells can migrate over

long distances to reach the amputation surface, (Stinson, 1964). If the

anterior part of a flatworm is irradiated and the posterior part shielded,

and then the head removed by a transverse cut within the irradiated region,,

head regeneration occurs with a considerable delay because the reserve

cells in the intact posterior region have to migrate across the irradiated

band to reach the amputation surface. The wider the band, the longer

the delay, but eventually a head is regenerated.

Metaplasia.

The question of whether metaplasia, as defined above, exists is 15 of great theoretical interest: it goes to the heart of the problem of cellular differentiation. Is the process of differentiation of an embryonic cell into a highly specialized cell~ such as a muscle or bone cell~ reversible?

can a full-fledged muscle cell revert to an embryonic state and then1 under certain conditions~ differentiate in a new direction and become a bone cell? The origin of the limb-regeneration blastema in salamanders

has always been in the centre of this discussion~ without1 however, yielding crucial evidence one way or the other. First~ the question had to be settled whether the blastema cells come from the amputation stump

or from more distant parts of the body1 or perhaps evenfrom formed elements of the blood. Irradiation experiments gave the answer. If part of a salamander limb is irradiated region, regeneration Jails to occur.

Conversely~ irradiation of the whole animal~ except for the limb1 does not interfere with limb regeneration. Contrary to what happens in

planarians1 then1 the blastema cells do not come from distant parts but are derived from cells near the amputation surface (Butler~ 1935; Butler

1 and 0 Brien1 1942; Brunst1 1950).

Microscopic study of the events following the amputation of a limb reveals that a dedifferentiation of differentiated muscle and cartilage or bone actually occurs near the cut surface during the few days preceding the formation of the blastema. The multinuclear muscle fibres break up

into fragments forming small 'cells with single nuclei1 which have the appearance of embryonic cells or fibroblasts (Becker~ 1961; Glade~ 1963). 16

There seems to be agreement that these cells, together with similar cells

of skeletal origin and with ordinary fibroblasts, contribute materially to

the blastema. If by metaplasia is meant merely the loss of highly spec-

ialized structure and the transformation into a fibroblast-like, embryonic-

looking cell, then this is a case in point. But this is not metaplasia in

the strictest sense. The crucial question is still open. Have these cells

actually lost all their biochemical and metabolic specifications? Have

they reverted to a true embryonic pluripotential state which would enable

them to differentiate into a cell type different from the one they repres-

ented before? A definitive answer to this question awaits new techniques.

There is, however, one clear case of true metaplasia. This is

the regeneration of the crystalline lens of the salamander eye, often

referred to as Wolffian lens regeneration in recognition of one of the ·1r. ~ .. co-discoverers and most active students of this very remarkable pheno- -'1 '::-.'·..'': menon. If the lens is carefully removed with fine instruments it is .

replaced by a new lens that originiates at the upper margin of the iris. ' The latter is the pigmented part of the eye, enclosing the pupil. The first change, following lens extirpation, is the disappearance of the

pigment in the upper iris; that is, a process of dedifferentiation. Next,

the two tissue layers that comprise the iris separate and expand at the .' t rim where they are continuous, and form a small vesicle. This vesicle grows downward to assume the normal position of a lens; eventually it

becomes detached from the iris and differentiates into a typical lens. 17

Here may be observed directly the transformation of pigmented iris cells into lens cells (Overton, 1965; Reyer, 1954; 1962}. Another case of true metaplasia, the regeneration of brain tissue from epidermis in annelids, is well documented (Eguchi, 1967; Yamada, 1963}.

Phylogenetic Survey of Regeneration--Invertebrates

Regeneration in Protozoa

Regeneration in protozoa is an interesting case in point. The animal is unicellular and regeneration must involve reorganization and remodeling of internal parts, but could conceivably occur without mitosis if there were no nuclear injury. Lund (1917} descirbed marked dediffer­ entiation of internal cell parts during regeneration in Bursaria. The ciliated adoral zone: mouth, gullet, cortical plates of cilia (membranelles) and cytoplasmic vacuoles decrease in size or disappear in fragments isolated by injury, just as they do in animals preparing to reproduce by fission. For regeneration to occur, part of the macronucleus has to be included in each fragmen~ but its size is unimportant. Redifferentiation during regeneration is essentially identical in the two cases. The de­ differentiation or simplification in structure of the macronucleus that occurs in both cases is presumably related to the replication of nucleic acids and proteins which must ensue. Interestingly, cells that become spores or cysts in adverse conditions must dedifferentiate before 18

redifferentiating into an active adult {Weisz, 1955).

Stentor exhibits physiological as well as reparative regeneration.

The mouth parts, for example, may be replaced from time to time

(Balamuth, 1940; Tartar, 1961). During reparative regeneration in

Stentor, oral structures always regenerate from the cut surface of the

posterior piece and foot parts regenerate from the cut surface of the

factors responsible may reside in the cortex, along the so-called left

boundary stripe.

After surgical excision of the anterior end, regeneration requires

about 12 hours in Stentor. It is essential that part of the macronucleus

be present. During the first 4 hours the kinetosomes (centrioles associated with cilea) divide repeatedly. Then membranelle synthesis ensues and

structural redifferentiation becomes apparent (Weisz, 1955). Bacter­

iostatic agents such as acriflavine prevent regeneration, presumably by

affecting the kinetosomes. The effect is counteracted by nucleic acids

and related compounds. Ribonuclease also prevents regeneration. This

effect is alleviated by ribonucleic acid (RNA). Since the production of new cilia is dependent on protein synthesis, the system might be exploited for an analysis of genetic control of morphogenesis. Preliminary studies of cilia regeneration in Tetrahymena indicate that deoxyribonucleic acid

{DNA) dependent RNA synthesis is required in some cases (Child, 1965).

Such cells would also be excellent for an electron microscopic study of the morphogenesis of cilia and centrioles (Williams, 1964). Cytological 19

and physiological studies of regeneration in the other protozoans as

well would be highly desirable. Most of the published work has been

carried out on ciliates and flagellates {Balamuth~ 1940; Weisz~ 1954;

Tartar~ 1961; and Berrill~ 1961).

Regeneration in Porifera.

Sponges display varying ability to regenerate after cutting. Those with a well-developed protective cortex are said to regenerate this part

poorly (Needham~ 1952). In ti1e invivo situation~ archaeocytes emerge from the gemmule, aggregate around it~ and differentiate into a new

sponge. In the in vitro experiments, the sponge is cut into bits that are

strained through fine bolting silk (H. V. Wilson~ 1907). The cells that emerge through the pores of the cloth reaggregate to form a new sponge.

Reaggregation is highly species specific. Recently, Humphreys (1963) studied cells of Microciona dissociated in calciumand magnesium-free sea water and concluded that the cells require the two divalent cations

(calcium, magnesium) and a cell surface factor, possibly a normal intercellular material which is the species- specific factor, in order to reaggregate at a temperature cold enough to prevent the synthesis of intercellular matrix.

Wilson (1907) concluded that the archaeocytes of basophilic mesenchymal cells of the sponge have the greatest regenerative powers of any of the cell types~ even though some of the others might undergo 20

"regressive differentiation into an unspecialized amoeboid condition"

under the conditions of isolation {Huxley~ 1911). The archaeocytes

also are said to give rise to new tissues in growing sponges~ in sponges

reforming from gemmules~ and in sponges regenerating after cutting.

"Totipotent" reserve cells of this sort have been implicated in regenera-

. tion in coelenterates~ flatworms~ annelids~ and certain tunicates, as

can be seen below.

Regeneration in Coelenterates

Various members of the three classes of coelenterates have

been studied and found to be capable of restoring complete organisms

from small fragments of the parents. Hydrozoa and Anthozoa, which

o·ccur as medusae and/ or polyps~ regenerate better than the Scyphozoa

{jellyfish). Some authors have considered that the interstitial cells are

reserve cells for all regenerative and budding process in coelenterates

{Lenhoff and Lommis, 1961: Berrill, 1961). The evidence for partici­

pation of the interstitial cells in Hydra regeneration stems mainly

from histological studies of normal regeneration. If a polyp is transected,

the edges of the wound are brought together by contraction of the inner

epithelium {the endodermis). The cells adhere to form a wound plug

which is covered within an hour by epidermis. Interstitial cells adjacent

to the wound seem to increase in size, divide, and migrate from

epidermis into the endodermis, transforming along the way into endo- 21 dermal cell types (gland or lining cells). Cell counts suggest that in both Hydra and Tubularia, the number of interstitial cells decreases in the rest of the body as the cells seemingly migrate to the wound area

(Tardent, 1963). In Tubularia, there is usually a rhythmical distal movement of cells along the stalk toward the regenerating area

{Steinberg, 1955). These consist of (1) interstitial cells migrating between epidermal cells and (2) endodermal cells moving as a group

(Tardent.~ 1963).

Further evidence for participation of interstitial cells in regeneration has been sought in experiments on irradiated animals.

Interstitial cells seem particularly sensitive to X-ray and will degenerate if the dose of roentgen rays is high enough. The deleterious effect of

X-ray on regeneration in coelenterates often is taken as evicence that interstitial cells are indespensable for regeneration {Burnett.~ 1962;

Tardent, 1963). There are, however.~ contradictory reports that Hydra can regenerate after irradiation, even though the interstitial cells are injured. Moreover.~ the possible effect of irradiation on other cell types usually is not taken into account in the interpretations of the inhibi­ tion of regeneration by effective doses of X-ray. X-rays could be expected to inhibit mitosis, not only of interstitial cells, but also of other cell types as well. Furthermore, the number of mitotic figures is said to remain low until redifferentiation has occurred in Tubularia.

If this conclusion is valid, it is difficult to understand why X- rays would 22 have any effect at all on early stages of regeneration {tardent, 1963).

On the other hand, Burnett {1962) reports evidence of cell proliferation within 2 hours of wounding in Hydra. The problem of mitosis and radia­ tion sensitivity obviously needs to be reinvestigated with newer techniques for studying cell turnover before the data can be applied in a meaningful way to an understanding of the origin of regeneration cells in Hydrozoa.

Haynes and Burnett {1963) have recently reopened the issue of the necessity of interstitial cells for regeneration in Hydra, using a different experimental approach. Taking advantage of the known fact that endodermis can give rise to epidermis in this animal, they selected a species, Hydra viridis, which lacks interstitial cells in the endodermis.

The endodermis alone contains the algal symbionts that impart the green color to the animal and therefore this tissue is provided with an excel­ lent marker for cell tracing experiments. The endodermis was isolated from the epidermis after treatment with trypsin by teasing the colorless outer epithelium away from the green inner epithelium. Even small fragments of pure endodermis regenerated partial or whole polyps. The mucous cells, zymogenic cells, and digestice cells of the endodermis became basophilic and seemingly dedifferentiated in part during the process. The endodermal cells transforming into epidermis lost the green algal symbionts in the process. Of considerable interest is the fact that new interstitial cells appeared in the regenerated epidermis 23 and that these endodermisderived cells gave rise to cnidoblasts just as would normal interstitial cells. Zwinlling {1963) showed that isolated ectodermal fragments of another hydrozoan~ Cordylophora~ could re­ constitute a whole animal. The eetoderm~ however~ contains inter­ stitial cells as well as epidermal cells in this animal and it could be argued that only the former participated in the cell transformations that occurred. Steinberg (1963) studied a scyphozoan, Aurelia, which lacked interstitial cells in the ectodermal areas selected for study.

She obtained good regeneration from siolated extodermal fragments which contained no interstitial cells. It will be recalled that in the hydrozoan, Limnocnida~ normal budding occurs in areas of the medusa which lack interstitial cells. Regeneration also can occur in Hydra from regions of the body in which interstitial cells are injured or absent

{Burnett, 1962; Diehl and Burnett, 1963). While a role in regeneration and budding cannot be denied to the interstitial cell~ its major function in the adult is production of cnidoblasts. The evidence on hand as to the participation of more differentiated cells in regeneration is so strong~ that it does not seem necessary any more to invoke the inters­ titial cell as a "reserve'' embryonic cell to account for regeneration.

Recognition of this fact calls attention to the need for a more detailed investigation of epithelial cytology during hydrozoan regeneration~ with the possibility that cell differentiation and proliferation in the formed tissues can be detected in all cases of regeneration in hydrozoans. 24

The phenomenon of polarity in regenerating systems has been

extensively investigated in hydrozoans, especially in Tubularia where

the process of regeneration can be followed very clearly in living

animals observed through a dissecting microscope. Tubularia occurs

in colonial clusters. The stalk (stem) of each animal contains a head

(hydranth) with a distal whorl of short tentacles and a second proximal

ring of long tentacles just below the gonophores. If the head is ampu­

tated, a broad zone of diffuse pigment appears in the endodermis

adjacent to the tip of the stem after the wound has healed. The pig­ mented area is the primordium of the new hydranth. More discrete proximal and distal pigmented bands delineating the future location of the tentacles are next seen. Definite proximal ridges that will give origin .to tentacles soon form. By the time the distal ridges appear, the proximal tantacles have started to separate from the stem.

Experiments combining proximal and distal parts of regenerat­ ing promordia in Tubularia are facilitated by the presence of an acellular perisarc around the stem, the speed of hydranth regeneration, and the availability of both red and yellow animals. Any region of the stem has the capacity to form any part of the hydranth, but it never reproduces that part which has already started to differentiate anterior to it (Rose, 1957}. If two like parts are joined at appropriate stages in regeneration, so that both face in the same direction, the one which is most anterior dominates development and tends to inhibit the proximal 25

part. The inhibitory information travels only from the distal to the

proximal end. If a graft of a distal part is put on the proximal end of

the host, the proximal host region forms a second set of long tentacles,

which has the imposed polarity of the graft (Rose, 1957). Experiments

by Child and others have demonstrated similar proximodistal "gradients"

· in other species (Child, 1941; Barth, 1940, Berrill, 1961).

Child explains polarity in terms of a single physicochemical

gradient passing from high intensity at the anterior end to low intensity

at the base of the animal. Double gradient hypotheses also have been

proposed (Tardent, 1963). Rose (1957) takes the view that differentia­

tion in Tubularia is controlled by inhibitor substances moving in a

distoproximal direction and he proposes a general theory of the role

of specific inhibition in development which ties together many observed

phenomena of this type. Numerous attempts have been made to isolate

and characterize such inhibitor substances in hydrozoans, but the

results have been inconsistent (Steinberg, 1954; Fulton, 1959; Rose,

1963; Tardent, 1963).

It is possible that the electrical differences in potential between

the anterior and posterior ends are responsible for movement of

"correlative" substances in animals. Indeed, applied electrical fields

can reverse the polarity of regenerating hydroids (Child, 1942; Lund,

1947; Barth, 1955). Rose {1963) found that an imposed current blocked

control by a distal graft when the distal end faced the cathode. He

30 identified by the oval or pearlike shape of the cells, the basophilic cytoplasm, and the well-developed nucleoli. In the normal animal. the number of mesodermal cells fitting this description diminishes from head to tail regions. Two days after decapitation. cell counts show that more basophilic cells are present near the sectioned sur­ face than normal for that area. Seemingly they derived from the prepharyngeal region which subsequently replaces its neoblasts with new cells believed to have derived by proliferation of neoblasts in even more posterior regions (Lender, 1962; Wolff, 1962; Stephan­

Dubois, 1965). The difficulty with this approach is that if a differ­ entiated parenchymal cell transformed to a basophilic cell type by dedifferentiation as suggested by Woodruff and Burnett (1965), it would have been counted as a neoblast in this experiment (Chandebois,

1965). The problem clearly needs further investigation with more clear-cut cell tracing techniques.

The distribution of regenerative capacities among the tur­ bellarians contradicts the popular assumption that all primitive animals regrow missing parts with ease. The capacity for anterior regeneration is restricted considerably in many of them. Members of the other flatworm classes, Trematoda (flukes) and Cestoda (tape­ worms), are said to lack regenerative capacity, but they have not been studied thoroughly. Acoela, worms which belong to the same class as planarians (Turbellaria), lack organs such as kidney and

33

Polychaete s (marine worms) are so named because of the numerous lateral tufts of setae (chitinous spines used in locomotion).

It is impossible to make a generalization about the regenerative

capacities of the various genera. AutelytuS1 which has been studied

by Okada (1929) and others, exhibits a clear-cut anteroposterior

gradient in regeneration. As in planaria1 the posterior regions

tend to produce smaller headS1 but a level is reached from which no head will form. In some species only the first two segments can

produce a head1 whereas in others the last three can reproduce

most of the body (Berrill1 1961). Growth from the hindpiece forward

" ·• is termed anterior regeneration. Growth from the amputated head-

piece backward usually is more limited1 but most worms regenerate well in this direction from any level posterior to the pharynx. In

Sabella, a new head forms from the cut anterior end of the abdomen and later certain parts of the abdominal segments metamorphose into thoracic parts. If the abdominal piece is cut posteriorly at the

same time so that it has two cut ends 1 then even more of the abdomen becomes thoracic in character. Rose ( 1957) explains these pheno-

mena in terms of changes in the polarity of the animal1 with corres- ponding differences in specific inhibitions of one region by another.

The class Oligochaeta includes the true e a number ~~'IJIS To of primitive aquatic forms~ and some inter ~"fe groups tltE"-1i LOYOLA \J\ in mud. In most oligochaetes~ and some pol chae~jV~}cryva1

/...iSRARY 34

growth is by elongation of individual segments that were laid down

earlier and are constant in number. Interestingly, posterior regene­

ration in these forms ceases when the total number of segments is

restored to normal (Moment, 1951, 1953). Lumbricus is incapable

of posterior regeneration and certain other earthworms can re­

generate only during the diapause which occurs annually. Anterior

_regeneration is limited to anterior regions in most earthworms, but

Lumbriculus can form a head from any level. In Perionyx millardi,

polar heteromorphosis tends to occur in regeneration from an

amputated headpiece. If the anterior head is removed later, the

newly formed posterior head becomes dominant and tail regeneration

ensues from the cut surface, thus reversing the original body polarity

(Gates, 1951}.

Posterior regeneration in the earthworm, Eisenia, has been

studied extensively by Moment (1953) who has formulated an electro­

motive theory to explain the controlling mechanisms. Moment found that the posterior tip of the worm is electropositive with respect to the rest of the body. The value becomes negative on amputation and

gradually rises again during regeneration. In this worm, the total number of segments is 100. If the amputation level is at segment 80, then 20 new segments form; at segment 50, 50 new segments form.

The acquisition of maximal electrical potential seems to be directly correlated with number of segments formed, not size of the segments. 35

Moment (1953) postulates that animals continue to grow by proli­

feration of voltage-producing units, until, by summation of these

units, a critical inhibitory voltage is built up. He calls attention to

"Morgan's law of regeneration" which states that proliferative growth

in all animals becomes more distal. Certainly, it seems clear that

bio-electrical fields do exist from one end of the worm to the other

(Smith, 1963) and that they may well exercise a control over re­

generation in annelids as well as in Nemerteans, coelenterates, and flatworms.

Judging from reports in the literature, the source of regenera­ tion cells in annelids is variable. In Euratella, posterior regeneration after irradiation is said to be accomplished by epidermal cells which proliferate to form bands of mesoderm and other tissues (Stone,

1933). In other annelids, new intestine is produced by growth poster­ iorly from the old intestine (Berrill, 1961). The new nerve cord probably arises from the epidermis as in Euratella. In Autolytus, the new mesoderm is thought to arise from dedifferentiated muscle.

In Nereis and Polydora, it is said that the ectodermal, endodermal, and coelomocytes are also believed to form the blastema in Nephtys.

The migratory cells from the coelom seem to attach to the surrounding ground substance when they reach the wound; then they lose their cytoplasic granules and take on the appearance of dedifferentiated mesenchyme (Clark, 1965). In other annelids, neoblasts are thought 36

to form the new mesoderm. In Lumbriculus, it is claimed that one

or two neoblasts normally lie on the ventral side of the coelom in

each segment and that they migrate posteriorly when activated. In

Tubifex, neoblasts are said to lie on the posterior face of the septa

separating segments; in Chaetopterus, between the pair of nerve

• cords (Berrill, 1961). While it is quite possible that there are real

variations among annelids as to the ability of formed tissues to de­

differentiate and the presence or absence of neoblasts, it is tempting

to believe that further study will reveal a common cellular mechanism

underlying blastema formation in all the annelids.

Regeneration in Arthropods

The jointed legs, chitinous exoskeleton, and specialized eggs

of the arthropods, especially the insects, have proved eminently

successful in allowing them to take to the land. There are 700, 000

species of insect, most of them terrestrial. The ability to regenerate

the appendages is lacking or incomplete in all of these many kinds of

adults (Needham, 1965). The life of the adult insect is often so short

that regenerative capacities seemingly would have little survival

value. There is no asexual reproduction. The methods of producing

eggs. however, are legion; in some insects parthenogensis is the rule

and there are no males.

During larval and pupal stages regenerative capacity is good 37

in the insects. Bodenstein ( 1955) has induced the adult cockroach to

regenerate a leg by causing it to molt. In one experiment a nymph

·is joined to an adult with an amputated limb. The adult molts as a

result of hormone supplied to it from the nymph, and the leg regene­

rates. The complex endocrine environment necessary for growth and

reproduction in the insect has been studied fairly extensively in recent

years (Krishnakumaren and Schneiderman, 1964; Wiggesworth, 1964).

In a recent review of the subject, Needham ( 1965) suggests that the initial phase of regeneration is controlled by juvenile hormone and does occur in the adult insect; the growth phase probably fails because of the lack of molting hormone (ecdysone). Nymphs can regenerate with an abnormal or incomplete nerve supply. It is possible, however, that minimal innervation of nonspeCific nature is needed. The nerves do regenerate readily and may have been present in small numbers in the material studied (Bodenstein, 1955; 1957; Penzlin, 1964).

Wolsky (1957) has studied regeneration of antennae in nymphs of the milkweed bug. The regenerates always had one segment less than normal regardless of whether amputation was between the second and third, or the third and fourth, segments. The heteromorphic re­ generates were often oversize. Wolsky explains this in terms of the unusual somatic polyploidy might have led to the abnormality of the regenerate. In any case, it is clear that a number of factors have to be taken into account in explaining growth and differentiation 38

in the insect. These highly successful arthropods have developed

nuclear and cellular specializations that are unheard of in other

animals.

The class Crustacea is a little more straightforward than the

Insecta. Lobsters, crayfish, and crabs continue to molt as adults

and so presumably do not lose the necessary endocrine balance for

growth and differentiation (Bliss, 1959; Durand, 1960; Needham,

1965). In fact, they seem to have capitalized on this feature in

evolving the phenomenon of autotomy. If the leg of a crab is seized,

it breaks off spontaneously at a preformed point across the second

leg joint by violent contraction of the extensor muscle of the leg.

The wound is covered with a chitinous plug and a new limb does not

grow out until the next molt. Chemical agents which irritate the

muscle can bring about autotomy of the legs. Morgan (1901) has

pointed out that even the abdominal appendages which are normally

sheltered in the hermit crab can regenerate. The case is used in

arguments against the adaptive significance of regeneration. A

unique kind of heteromorphosis called homeosis occurs occasionally

in arthropod regeneration. The classical example is the formation

of an antenna instead of the normal eye stalk when the latter is

amputated in the shrimp (Morgan. 1901; Needham, 1965).

The origin of cells in regenerating crustacean limbs has

been the subject of controversy (~eedham, 1952; 1965). Regenera­ tion in continuity is impossible after autotomy. Morgan (1901) 39

believed that the new muscles formed by dedifferentiation of ecto­

derm and he capitalized on this point to argue against the specifi­

city of the germ layers (Schotte~ 1940). The alternative to a local

origin of cells from the epidermis is migration of cells with the

ingrowing vessels and nerves. Since the developing muscle inserts

. on the exoskeleton, it must pierce the epidermis impression that

epidermis is transforming into muscle (Needham, 1952; 1965).

Regeneration in Echinoderms

Whereas the artropods and annelids have evolved along the

main line of protostomes, echinoderms represent an evoluationary

branch of deuterostomes which seems to have led directly to the

chordates. Endoskeletons, absent in protostomes, are common in

deuterostomes. The echinoderm has a calcareous skeleton which is

formed by mesoderm in the deep layer of the skin. The watervas­

cular system is unique. The middle part of the left coelomic sac

forms a ring around the esophagus and develops blind, radial canals

from which the tube feet arise,

Starfish and brittle stars can regenerate arms from the

central disc and can reform a whole animal from an arm, if part

of the central disc is attached. Sea urchins can repair damage to

the skeleton and tube feet. The sea cucumber responds to certain

external stimuli by eviscerating the alimentary canal and other 40

internal organs. The remaining shell of skin and muscle is capable

of regenerating the autotomized organs. Certain sea cucumbers

and starfish fragment at intervals to produce new individuals by

asexual reproduction (Berrill, 1961; Hamburger, 1965).

The larvae of echinoderms do not seem to exhibit very great

powers of regeneration nor do the larvae of annelids and tunicates.

The larvae of Arbacia, which live about 4 weeks, do regenerate

their arms. Adult echinoderms and annelids live longer than the

larvae and, in general, have better capacities for regeneration.

Needham (1952) suggests that regeneration probably is evoked too

rarely in ephemeral (short-lived) forms to have survival value and

therefore does not exist. This view seems overly teleological,

however. Regenerative powers which have no survival value are not

uncommon among animals, and it would not be surprising to find

greater regenerative capacities among the echinoderm larvae, were they studied more thoroughly.

Anderson ( 1965) recently has extended his cytological studies of regeneration in echinoderm adults to include autoradiographic data on incorporation of tritiated thymidine. His article, and the

book by Hyman (1955), serve as good introductions to the available literature on the origin of regeneration cells in this group.

Anderson's earlier belief that mobilization of amoebocytes is re­ sponsible for regeneration of the caecum in sea stars was revised in 41

the light of his autoradiographic studies which demonstrate con­

siderable DNA synthesis in cells of the lining epithelium and

covering peritoneum at all levels of the regenerate. For most of

the other echinoderms. cytological data are not very clear cut.

In holothuroids (sea cucumbers). it would appear that the anlage of

. the new gut derives from a solid cord of mesenchyme. Growth

henceforth is by mitotic activity in the gut lining and the new layers

are continuous with the old. In Stichopus. it is said that the lining

of the gut proliferates and differentiates from mesenchymal aggre­

gations without continuity of layers.

Regeneration and Budding in Tunicates

The Tunicata or Urochordata comprise one of three chordate

subphyla. Their regenerative powers are truly remarkable, surpass­

ing the subphyla Vertebrata and Cephalochordata. and the related

phylum Hemichordata (Needham. 1952; Berrill. 1961; Tweedell.

1961). The ascidians are found along most shore lines and have been

studied in more detail than the oceanic classes of tunicates.

Ascidian eggs develop in typical chordate fashion, but they

metamorphose into saclike animals that bear little resemblance to

adult vertebrates and cephalochordates. As larvae. tunicates have a

dorsal notochord, muscles, and a tubular spinal cord. At metamor­

phosis, the tadpole attaches to the sea bottom on its nose and the 42

tail is resorbed by a remarkable process of cellular dissociation

and migration. The epidermis then secretes an external tunic

which contains collagen, cellulose, and a few cells. The barrel­

shaped pharynx dominates the internal organs of the adult. Water

enters through the branchial siphon and passes through gill slits

. in the vascularized pharyngeal wall into a peribranchial chamber

that is connected to the exhalant siphon. The digestive trace ex­

tends from the lower end of the exhalant siphon. A glandular endo­

style, neural mass, ovary and testis, heart, and circulatory system

complete the structural complex.

Regeneration and asexual reproduction by budding are closely

related processes in the ascidians. The epidermis, while highly

differentiated and never itself seeming to transform into another

tissue, may play an important role in the process. It is clearly the

agent that isolates the fragments (Berrill, 1961). The inner tissues

of the bud derive from different sources in different species: (1) the

epithelium lining the atrial or peribranchial cavity, as in Botryllus;

(2) the lining of the epicardium, a cavity adjacent to the heart; (3)

the mesodermal septum separating afferent and efferent blood flow,

for example, in the stolon of the colonial tunicates, Clavelina and

Perophora. Participation of other organs has been described and

attention has recently been called to a possible role of blood cells

in budding in Perophora (Freeman, 1964). 43

Budding in Botryllus is part of a delicately timed reproduc­

tive process which involves simultaneous development of gametes

and regular, precise dissolution of the parent zooid. Maturation of

the gonads coincides with full development of the bud, fertilization

occurs, an embryo develops in the peribranchia cavity, and the parent zooid degenerates at a predetermined time. In the meantime, a second generation of buds has arisen in the zooid. Premature dis­ solution (as by injury of the parent) simply speeds up maturation of buds. If buds are extirpated, the survivors grow more quickly. Re­ generation, then might be said to be compensatory in nature in

Botryllus.

Clavelina forms colonies of zooids connected by a stolon partitioned by a mesodermal septum into two vascular cavities. At the end of the breeding season, only the stolon is held over for the following spring and it will form the new buds that reproduce the colony. Pieces of stolon cut during this formative period regenerate from the cut ends, but later the cut ends merely heal over and new zooids are formed by compensatory regeneration at pre -established budding sites along the stolon piece. The septal tissue forms the inner vesicle that gives rise to the organs other than epidermis in the new bud. The body of the zooid is also capable of regeneration.

It it is amputated below the thorax, a new thorax forms from epicar­ dium and unites with the esophagus. Under certain conditions zooids 44

of Clavelina during resorption form restitution bodies. All of the

specialized cells regress, seemingly leaving the unspecialized epi-

cardial tissue which is said later to proliferate and to reform new

internal organs {Berrill, 1961).

In another ascidian, Perophora, which also forms colonies on a vascular stolon, certain chemicals cause the zooids to regress and the organs seem to dedifferentiate to give rise to simplified, spheroidal cells which migrate back into the stolon {Huxley, 1921).

The zooids also regress completely if they are injured by a cut.

Later, new zooids arose from blood cells and dedifferentiated somatic cells or was growth entirely by septal proliferation as re- ported in normal budding? The stolon is quite transparent and the migration of cells from the cut zooid and their accumulation in the underlying stolon is easily visualized. In the summer of 1959 in

Woods Hole, Massachusetts, Hay repeated Huxley's experiments on

Perophoraviridis. Pieces of stolon with one or more zooids were isolated in dishes and one zooid was amputated through the thorax or injured with a needle. The amputated zooid of the group described here contracted and maintained some circulation for a day. Three days later, the so-called dedifferentiation was complete and the underlying stolon, usually transparent, was full of green cells from the zooid. Soon, the cut end of the stolon nearest the accumulated cells began to grow. Within 2 weeks, a pair of new buds made their 45

appearance on the stolon and all traces of the old mass of cells dis­

appeared. Either the cells derived from the injured zooid or their

nutritious by-products gave rise to the new growth, for the animals

were not given a change of water and had no access to food. Stolons

which did not seem to receive new cells from old zooids did not pro­

duce new zooids under the conditions of these experiments. Since

the point of the work was to trace the fate of the old cells., zooids

were labeled with tritiated thymidine and transplanted to unlabeled

colonies. Some transplanted to unlabeled colonies. Some transplants

took and regressed into the stolon after injury., but autoradiographs

were unsatisfactory. Nevertheless, the feasibility of labeling

ascidian tissues with isotopes and following the cells is attested to

by the experiments of Sister Florence Marie Scott (1963) on Amaroe­

cium. She was able to show by autoradiography that during the re­

markable reconstitution of zooid that occurs in this species, the

minced tissues inserted into a common tunic reaggregated like­

tissues from like-tissues.

Certainly, there is very good reason to study these reproduc­ tive phenomena further in ascidians. as Barth (1955) has emphasized.

Clavelina is easily obtained in Florida and the zooids are 2 crp long, as compared with 2 mm for Perophora. The ability for modulation exhibited by certain internal areas in ascidians, contrasted to the · apparent rigidity of the epidermal cells, may have a bearing on the 46

behavior of vertebrate cells. The important thing to realize, which

seems to have been overlooked in choice of material for regenera­

tion studies in the past. is that this group of invertebrates is more

closely akin to the vertebrates than any other group which has been

studied. It often is speculated that a primitive deuterostome related to an echinoderm larva gave rise to the chordates and, in one way or

another, to the vertebrates. It is equally possible, as Berrill has

suggested, that the larva was secondary; that some remote sessile tunicate, with a dorsal nerve net, produced the first larva with a dorsal spinal cord; that these cells, in these strange marine crea­ tures, are more closely related to our own in behavior and genetic control mechanisms than to any cell in an insect, flat-worm, or coelenterate.

Vertebrates

Regardless of the likely possibility that the vertebrates de­ rived from the same primitive stock as did the echinoderms and urochordates, it is quite clear that they are a very different kind of creature in the present state of evolution. Asexual reproduction has been completely abandoned by the animals comprising the subphylum

Vertebrata and a terrestrial habitat has been assumed by many. It is not known whether or not the primitive vertebrates possessed great regenerative powers. The immediate progeny of the ancestral 47

vertebrates are, of course, animals which have undergone further

evolution. The larvae of Petromyzontes, the so-called ammocoetes,

are said to regenerate the tail (Niazi, 1964), and members of the

superclass Pisces can regenerate the bony fins, optic nerve, and

taste barbels (Nicholas, 1955; Goss, 1956; Hass, 1962). The fins

cannot be cut too close to the body or they fail to regenerate. The

anal fin of the male Platypoecilus loses the capacity to regenerate in

the adult, an irreversible loss which can be induced in the female as

well by early treatment with androgens (Grobstein, 1947).

The salamanders seem unquestionably to have the most re­

markable regenerative abilities of all the vertebrates. With the

possible exception of a few species (such as Xenopus laevis), regene­

ration capacities seem quite limited in the adult anurans, yet these

amphibians are more closely aligned with the main stem of evolution

that gave rise to reptiles than the side-branch urodeles evolved new

regenerative abilities as to make any claim that they retained some­

thing the higher vertebrates lost. In fact, the distribution of regenera­

tive capacities among the urodeles supports such a speculation (Rose,

1944). Mechanisms for regenerating the lens are quite different among the salamanders. One family has the capacity to regrow the whole eye and optic nerve from the pigmented epithelium of the retina.

Some adult land salamanders are said to regenerate the appendages

Well (Plethodon), others regenerate poorly (Ambystoma) and there is 48

variability in regenerative capacity among the aquatic forms. Inter­

estingly enough, a genetic mutation in the axolotl, which prevents

this neotenic aquatic salamander from regrowing a limb, has been

observed (Humphrey, 1966). It is tempting to think that a decrease

in relative numbers of nerves accompanied by an increased thres­

hold to the trophic action of the nerve is one cause for failure of

limb regeneration among the higher vertebrates (Singer, 1952; Zika

and Singer, 1965). Other possibilities will be considered at the end

of the chapter, but it should be remembered that these comparisons

are between the modern frog or higher vertebrate. Nothing is known

of the regenerative capacities of our prehistoric ancestors.

The frog larva is capable of regenerating the tail and hind

limbs (Coulombre and Coulombre (1965). The capacity to regenerate

the hind limb is lost at metamorphosis, with the proximal parts

losing the ability before the distal ones. This response can be brought

about by the hormone of metamorphosis, thyroxine is not the "cause."

The cause is the structure and function of the adult limb, its in­

nervation, the controlling mechanisms for growth in these animals,

and, ultimately. the genetics of metamorphosis. The effect is

permanent, even if the thyroid is later removed from the animal.

Embryos and young animals often are said to possess greater re­ generative capacities than adults. There are so many exceptions to this statement, however, that it can hardly be called a rule. The 49

transition which occurs from larva to adult during metamorphosis

in the frog is best treated as a special phenomenon within that

biological order.

The reptiles derived directly from stem amphibians along

the main line of evolution, the most important advance being the

acquisition of the terrestrial (amniote) egg. They have limited re­

generative powers as compared with the urodele and frog tadpole,

but nevertheless have evolved some interesting mechanisms which

involve regrowth of body parts. The Lizard discards its tail by a

process of autotomy not unlike that whi~h has evolved independently

among the crustaceans. The regenerated tail, however, is far

from perfect. Nerve and muscle are atypical and the cartilagi­

nous axial skeleton does not segment or ossify (Woodland, 1920;

Barber, 1944; Kamrin and Singer, 1955; Simpson, 1965). The em­

bryonic lizard does not have the ability to regenerate the tail

(Moffat and Bellairs, 1964).

Reports of regeneration among the birds and mammals empha­

size the variability of the process. Physiological regeneration is

quite well developed. Epidermal appendages, such as feathers, nails, and hairs, either grow continuously or are replaced by per­ iodic molts, as was also probably true of the scaly epidermis of our immediate terrestrial ancestors. Regeneration of functional axons in the central nervous system is better developed in birds than in 50

mammals and perhaps is better in embryos than adults (Windle,

1955). Mammals have a remarkable ability for liver regeneration.

In fact, this particular capacity seems better developed in higher

vertebrates than in salamanders (Bucher., 1963; Grisham, 1962).

The salamander, because of its dramatic regenerative re-

. sponses, has been the subject of much study in the past. The re­

generation of the lens, retina, and optic nerve, and the regrowth

of the limb have been demonstrated. This does not mean that

other parts of the body have not been studied. Nor are they less

interesting. Regeneration of the urodele tail, bony vertebra, and

spinal cord, but not of the notochord, occurs readily (Piatt, 1955;

Holtzer, 1959). There is no direct evidence that differentiated

nerve cells can divide. In the regenerating spinal cord of the uro­

dele larva, cells are "paid off" from the transected gray matter to

cross the ablation gap; the neuroblasts that give rise to new motor

neurons may have derived from epndyma (Butler and Ward, 1965).

Goss and Stagg (1958) report that the newt jaw regenerates after com­

plete amputation or excision of the central portion, if enough mandi­

ble is left to support the process. Regeneration of the intestine is

excellent in both frogs and newts (O'Steen, 1958; 1959). The loose

ends find each other and rejoin end to end or side to side. The

blastema forms by dedifferentiation, but during redifferentiation the

cells are believed to sort out according to their origin (0' Steen and

Walker, 1962). 51

Mammals

These present numerous instances of tissue regeneration,

but· very little, if any, evidence for the regeneration of complex

structures. The change of hair of teeth and of red blood cells and

the regeneration of antlers in deer illustrate physiological re­

generation. Reparative regeneration, following an amputation is

limited to tissues, such as bone, muscle, liver, skin and peri­

pheral nerves. Whole organs cannot be regenerated.

Every vertebrate is dependent to some extent on physio­

logical regeneration for survival and in no case is this better exem­

plified than in the mammal. One of the distinguishing features of the

class is that the members are covered with hair which is periodi­

cally molted. The surface epithelium is a stratified epidermis

specialized for continuous replacement of cells in the outer, protec­

tive layer. A cell in the basal stratum of the multi-layered squamous

epithelium of the mouse tongue may reach the surface in a little over

a week. Most of the skin appendages, such as hairs, sebaceous

glands, nails, claws. hoofs, scales, and horns, grow continuously or periodically. The antlers of deer, which consist of bone covered ini­ tially with a hair skin, are shed annually and replaced by new growth.

The dermis, sometimes considered to be the tissue that inhibits-limb regeneration in mammals, does in fact play the most important role 52

in bringing about regeneration of the antler (Goss1 1964a). Teeth~

which evolved from placoid scales such as occur in sharks~ are lost

and replaced continuously in lower vertebrates~ but in mammals they

are more specialized and are shed once1 then replaced by a perma­

nent set. If the roots remain open, however1 the teeth may continue

to grow for the life of the animal (rodents).

The renewing surface epithelia of the body are capable of re­

pairing wounds of considerable size after a variety of accidents~ such

as burns~ abrasions and cuts. The process of epidermal healing when

the skin is cut or surgically excised will be considered below under a

separate heading. The corneal epithelium has the ability to migrate

very rapidly over a denuded surface. Homografts of cornea take very

well because of the lack of vascularization and immune response and

are eventually repopulated by cells of the host. In mammals such as 1

the rabbit1 lens epithelium has some ability to regenerate (Harding and

Srinivasan, 1961). The regenerative ability of the urinary bladder,

gall bladder, and intestine have been demonstrated repeatedly (Goss,

1964). The type of healing and proliferation that occurs periodically during the menstrual cycle in the primate might be classified as re­ parative as well as physiological regeneration. A Number of hormones and complex controlling mechanisms are involved in the cyclic pro­ cesses.

The ability of the hemopoietic tissues to regenerate has been 53

used to advantage in modern surgery. It has been possible to trans­

plant organs# such as kidney# between genetically different individuals

of the same species (homografts). In the pioneering work along these

lines, hemopoietic tissue of the host was suppressed prior to the

operation by total body irradiation, with the idea that all of the new lymphoid cells would develop in the presence of the graft. The re­

generated cells# like embryonic blood cells, would be expected to be tolerant to the foreign graft, so that fatal immune responses are avoided (Billingham, 1964). Currently, drugs rather than irradia­ tion are more often used to suppress the immunological response after surgery employing homografts (Russell and Monaco, 1964).

Among the so-called stable group of cells# the ability of epithe­ llal glands to regenerate is particularly notable. The liver of the rodent, which normally is growing to some extent# has been investi­ gated most extensively (Bucher# 1963; Grisham, 1962; Goss, 1964), but it is clear that the human liver, too, has remarkable powers of recuperation. If the liver is massively damaged or partially excised

(three quarters of the rat liver can be removed), the first observed change in the remaining liver cells is an increase in RNA and protein synthesis. Enzymes (for example, thymidylic kinase) related to DNA synthesis make their appearance. The cells might be said to have entered the G 1 phase of the progenitor cycle. In the cast of rat liver,

30 percent of the remaining liver cells have begun to synthesize DNA 54

at 20 hours. At 26 hours~ the mitotic index reaches a peak ( 3. 6 per­

cent of the cells are in mitosis).

Proliferation soon slows~ presumably under the influence of

humoral factors~ so that by 3 days only 3 percent of the cells are

synthesizing DNA (Bucher, 1963; Grisham, 1962; Shea~ 1964). So

· rigid are the control mechanisms, that the liver never overshoots its

original size during regeneration (see discussion of compensatiory

hyperplasia by Goss, 1964b). Interestingly enough~ the proliferating

liver cells do not seem to dedifferentiate. Presumably, the period of

maximal cell division (1-2 days) is attained so readily and is so short

in duration, that the rather stable differentiated cytoplasmic organ­

elles and ribosomes of the liver cell persist in spite of the fact that

metabolism has shifted temporarily to DNA synthesis.

The fibrocytes, chondrocytes, and osteocytes that secreted the

connective tissue matrix are, in most mammals, rather quiescent

after the animal reaches maturity. The nucleus of the cell is usually

fairly condensed and the cytoplasm is scanty or empty appearing. In

order for significant growth to ensue, the tissue must dedifferentiate

in the sense that a certain amount of the confining extracellular matrix

must be lysed and new machinery for synthesizing nucleic acids and

proteins must be acquired by the cells (Goss, 1964). If a bone is

fractured, for example, the following series of events takes place.

Soon after the fracture, there is a certain amount of cell death in the 55

broken ends of the bone and the surrounding tissue due to the interrup­

tion in the blood supply. These cells will be lysed and the matrix

that surrounded them will eventually be resorbed, as will the blood

clot that usually forms around the bone fragments after injury. With­

in 2-3 days, cells released from the periosteum endosteum, and

• perhaps also from the marrow and adjacent connective tissue, begin

to proliferate. They form a collar or callus around the ends of the

bone which serves as a natural splint to protect the injured site. The

length of time required to form the cellular callus, its size, and the

extent to which the callus chondrifies prior to ossification depends on

the size of the injury, the type of surgical intervention, and other

factors (Ham, 1965). The callus is usually chondrified and partially

ossified within 2 weeks. Remodeling of the newly formed bone may take years.

Cell dedifferentiation after injury is perhaps most marked in muscle which (Thornton, 1938), it will be recalled, consists of static cells. The developmental processes that led to the formation of the multinucleated (syncytial) striated muscle fiber seem to be incom­ patible with proliferation. An injured muscle fiber always loses cyto­ plasmic structure and tends to fragment into smaller cellular units.

The extent to which the mononucleated cells derived from the fibers are able to divide and join up with similar cells on the other side of the lesion determines the success of the repair. Thus, if the ends of 56

severed muscle fibers are closely juxtaposed, muscle regeneration in

mammals can be excellent. But if the severed ends are separated,

the space will fill in with connective {scar) tissue. In the case of

cardiac muscle, not manifested to a great extent because the injury is

usually ischemia. The cells are deprived of the necessary metabolic

conditions for growth. Repair processes in smooth {nonstriated)

-muscle have not been fully clarified in mammals, but in amphibian

wound healing, dedifferentiation is involved. The smooth muscle of

the mammalian uterus is capable of considerable expansion, perhaps

due more to cellular hypertrophy than to hyperplasia. In healing after

surgical incisions of the uterus, however, new smooth muscle cell

formation has been reported (Selye and McKeown, 1934).

The neuron, like the myocyte, is a permanent or static cell

type in the normal adult. Even though the cells seemingly exert a

trophic action on the growth of other tissues, neurons themselves have

no capacity for cell division. The ability of the mammalian neuron to

regenerate axons is confined to the peripheral motor and sensory pro­ cesses. Central neurons are reported to send out cytoplasmic process­ es after injury to the spinal cord, but these axons do not grow far enough to establish functional continuity within the cord. Failure of complete regeneration of axons in the mammalian central nervous system may be due to the complex arrangement of the neuroglial sheaths. The Schwarm cell sheaths that occur around peripheral axons, 57

as we shall see below, have an important role in successful regenera­

tion of these fibers. Interestingly enough, the neurosecretory function

of the hypothalamic tract to the pituitary can be restored after tran­

section (Hild and Zetler, 1953).

The ability of sensory and motor nerve cells to regrow their

. long peripheral processes, which are often a yard or more in length,

attests to the considerable synthetic capacity of these enormous neu­

rons. In spite of (or because of) its irreversible commitment to a

life without mitosis, the neuron is an exceedingly active interphase

cell. The nucleus is large and vesicular, and the nucleolus is promi­

nent. Ergastoplasm and free ribosomes are abundant in the cytoplasm

and the Golgi complex is well developed. The membranous organ­

elles probably are involved in neurosecretion, whereas the free

ribosomes most likely are synthesizing new proteins to replenish the

cytoplasmic processes. Proteinaceous materials flow down the axons

at the rate of 1-2 mm a day. A regenerating fiber grows at about the

same rate. The flow of protoplasm down the axon can be demonstrated

by constricting a regenerating fiber after it has made its connection

with the periphery. A bulge will appear on the proximal side of the

constriction and there will be no further increase in width on the

distal side (Weiss and Hiscoe, 1948).

When a peripheral nerve fiber is crushed severely or cut, the

axon distal to the injury degenerates completely (Stone, 1959; Reyer, 58

1962). Proximal degeneration of the axon rarely extends past the

nearest node of Ranvier. The cell body of the affected axon reacts to

the injury in a characteristic fashion. The Nissl bodies undergo

chromatolysis within 24 hours, that is, the cytoplasm loses the

typical basophilic staining reaction, or "color. " This "retrograde

degenerative process" reaches its maximum in a few days and results

in the loss of considerable ergastoplasm. The nucleus and residual

Nissl bodies take up an eccentric location in the injured neuron. The

nucleolus increases in size and presumably new ribosomes are formed

which take part in the protein synthetic processes that must occur if

cytoplasmic regeneration is to take place. The neuron does not re­

sume its former state of differentiation for a number of weeks. The

distal axon, and myelin sheath if present, have broken up into frag­

ments and droplets during the first few days after injury. This process is called Wallerian degeneration. The Schwann cells exhibit acid phosphates activity and probably take part in the phagocytosis of lysed debris (Stone, 1959).

As early as 4 days after injury to the nerve, new pseudopodia appear at the cut ends of the axons. They ramify through the sur­ rounding debris, guided by the Schwann cell sheaths, seemingly moving along the lines of least resistance in the external milieu (contact guidance theory of Weiss, 1955). If the injury is extensive, surgical intervention to join the ends of the nerve is required. Otherwise the 59

fibers will form a useless, sometimes painful neuroma, for lack of a

clearcut path to follow. If a motor fiber by mischance grows down a

sensory fiber Schwann sheath, restoration of function does not occur

very readily even when sensory or motor fibers make smaller errors

than this. The total repair process may take months, depending on

the problems encountered by the growing nerve fibers (Edds, 1953).

Fishes

If Nobel prizes were awarded to experimental animals instead

of the people who work on them, fishes would be the more richly de­

serving laureates in recent years. Scales, tail fins (Morgan, 1902;

Birnie, 1934; Comfort and Doljanski, 1958; Goodrich and Green, 1959;

Goss and Stagg, 1957); barbels (Goss, 1954; 1958; Kamrin and Singer,

1955), have been proven to regenerate.

Few objects in nature rival the exquisite delicacy of fish scales. From the microscopic sculpturing on their surfaces to the prevision of their arrangement on the body, the scales of fishes offer many a challenge to the curiosity of the biologist. One of the inter­ esting things about many kinds of fishes is that their growth is without apparent limit--like the stories fisherman tell about them. Since their bodies can continue to increase in size throughout life, while the number of scales remains fixed, it is up to the individual scales to keep pace with the systemic growth by enlarging proportionately

(Goss, 1956). Scales have not only been proven to regenerate but it 60

is also easy to transplant.

It is easy to transplant scales. The scale pocket in the skin is

a ready-made graft site into which the same or a different scale of

appropriate size can be inserted. If care is taken not to disturb the

fish unduly after the operation, the graft soon grows in place and may

re-establish its vascularity within a day or so. Homografts, which

come from other individuals, are rejected by the host, sometimes in

only a few days depending on the temperature. If the transplanted

scale has pigment cells in its adherent skin the first signs of graft

destruction can be detected by the abrupt breakdown of these cells;

this does not occur autografts, which are derived from the same fish

(Goss and Stagg, 1957).

Transplantation is a useful technique for studying the regenera­

tion and morphogenesis of scales. For example, if a scale of one

kind is substituted in place of another, will its morphology be altered

according to its new location? The lateral line scales of the goldfish

have holes in them through which the lateral line canal passes. When

plucked, their replacements are likewise perforated by the regenerat­

ing extension of the canal. If an ordinary scale from elsewhere on the body is transplanted in lieu of a lateral line scale, it too develops, a hold after a month or two. Apparently the scleroblasts possess morphogenetic potentials epecific for their native locations on the body surface. It also seems that any scleroblasts accompanying the 61

transplants are of little or no value in maintaining the morphological

integrity of the scale (Goss and Stagg, 1957).

The latter conclusion is substantiated by further experiments

carried out on the killifish (Fundulus heteroclitus) in which scales

have been transplanted to regions of the body other than the scale

pockets. When grafted subcutaneously to the fin, or inserted into the

eye beneath the cornea, scales invariably undergo erosion, presum­

ably due to the paucity of scale-forming cells. Scales, therefore, are

totally dependent upon the integumentary pockets in which they reside

for their maintenance and growth. It might be pY'edicted that scales

themselves cannot regenerate but that populations of scleroblasts can

(Neave, 1940; Oosten, 1923).

The ability to repair defects inflicted on scales can be in­ vestigated only if they are put back into skin pockets lined with

scleroblasts (Goss, 1957). After a suitable length of time such scales can be removed for examination. When scales are bisected trans­ versely or longitudinally and only one half is replaced in the scale pocket, the missing proximal or lateral halves are replaced adjacent to the residual parts of the scales. If half of the scale pocket itself is cut away after the scale has been plucked, the remaining half re­ generates only half a scale. It does not form a whole scale of miniature proportions. The scleroblasts are therefore arranged in a mosaic pattern which is capable of little or no regulation. Within the 62

scale pocket each cell or group of cells is responsible for producing

its own part of the scale and cannot change its job for the sake of the

organization (Goss6 1956).

Fin Regeneration

Fishes have evolved various kinds of locomotory appendages

which differ from one another in their skeletal support and their

capacities for regeneration. The fins of sharks, for example, are

supported basally by cartilaginous elements and distally by horny

ceratotrichia. They cannot regenerate (Goss and Stagg 1957). In

the African lungfish, the fins are slender tapering appendages com­

posed of long chains of cartilaginous articulations. These "fins" do

regenerate after amputation, and have been known to produce side

branches from lateral lesions. Teleosts possess fins made up of

ossified plates in their proximal regions which articulate distally with the rays (lepidotrichia) of dermal bone. These fins regenerate excellently (Conant, 1970).

The phylogenetic origins of the teleost fin and its rays have long been the subject of speculation among biologists. It has been proposed that the bony rays might be homologous with scales. since scales are lacking on the fins of teleosts (but are present on elasmo­ branch fins which do not have bony rays) (Goss, 1957). Histologi­ cally, the dermal bone of rays and scales is similar, at least to the extent that it is often acellular (Goss, 1957). But if rays did evolve 63

from scales, is each entire ray equivalent to one scale or to many of

them? In the former case, it is conceivable that the ray segments

might be homologous with the growth rings of scales, a possibility

not without some appeal in view of how fins grow (Goss, 1957).

Like scales, fin rays grow by addition. They add to them­

selves terminally because the ray segments already formed cannot

elongate. Fins therefore possess a generative zone along the outer

margins which provides for their unlimited growth commensurate

with the increasing size of the body as a whole. At the very end of

each fin ray is a tuft of actinotrichia, which are fine hairlike struc­

tures of uncertain composition located in the connective tissue matrix.

First produce in the natatory folds of the embryo, the actinotrichia

persist throughout life where ray development occurs. Although

they precede the bony ray in time and space, their significance is a

mystery (Goss. 1967). One can only surmise that they may serve to

support the soft tissues at the end of the fin until the rays become

ossified.

Teleost fins are compound organs made up of numerous fin

rays. Each ray is segmented throughout its length and branches

dichotomously as it elongates. The rays are actually double struc­ tures, consisting of paired components beneath the skin on either side of the fin. Between these two halves of the ray are sandwiched nerves and arteries, and between adjacent rays there are veins em bedded in 64

the soft tissues of the interradial zones. Amputation proximal to the

basal articulations of the rays seldom if ever leads to regeneration.

Distal to this level, fins readily replace missing parts by virtue of

outgrowths emanating from the stumps of the rays. Fins do not re­

generate laterally, but if amputated on the bias the rays tend to grow

out at an angle, more or less perpendicular to the cut surface. In

general, the fin regenerates as a whole only because each one of its

component rays can regenerate as an autonomous unit (Goss, 1957).

After healing has closed the wound on an amputated fin, a

blastema develops by the accumulation of apparently undifferentiated

cells derived in part from the loose connective tissue of the radial

and interradial regions, and in part from osteoblasts associated with

the ray stumps themselves (Conant, 1970). The latter cells can be

distinguished by their greater cytoplasmic basophila. They aggregate

off the ends of the rays and later become intimately associated with

the epidermis over-lying the blastema. It is at this point of juncture,

in the basement membrane between the epidermis and the osteoblasts

beneath it, that the earliest signs of new ray formation can be detected.

The new fin rays thus develop initially along the two sides of the elongating regenerate, and only secondarily do they become contiguous with the old ray stumps located proximally at the level. of amputation.

As regeneration proceeds, the newly forming rays acquire segmenta­ tion at regular intervals along their lengths. Actinotrichia are present 65

at the distal ends of the regenerating fin rays, as was the case in the

ontogeny of the original fin, (Goss and Stagg, 1957).

One of the more conspicuous attributes of the fin blastema is

the basophilic staining quality of the cells, particularly those engaged

in ray regeneration (Sichel, 1962). Presumably the cytoplasm of

these cells is rich in ribosomes, and their future differentiation may

well depend upon the RNA they contain. This hypothesis has been

elegantly supported by the research of Giovanni Sichel at the Univer­

sity of Catania in Sicily. In the monnow Gambusia, he has shown that

daily injections of ribonuclease into the abdominal cavity suppress the

regeneration of the amputated tail fin. This confirms the prediction

that blastema cells should be unable to differentiate in the absence of

sufficient RNA to make the required proteins, and that unless cells

differentiate organs cannot develop (Tasaawa and Goss, 1966).

In contemplating the course of events in fin regeneration, one wonders to what extent the blastema depends upon the tissues in the

stump to determine what it shall become. The bony fin rays, being the dominant formed elements in the appendage, are obviously potential sources of morphogenetic information for the regenerate.

If one of the two halves of a ray is entirely removed from the fin its counterpart in the regenerate does not develop. Such a fin grows out with only the half of the ray corresponding to the one left intact. How­ ever, if the ray is not completely excised so that part of its stump 66 remains behind, then regeneration commences at that level and only later does it catch up with the blastema that forms farther out where the fin as a whole may have been amputated. It can be concluded, therefore. that ray regeneration cannot proceed unless part of the original ray is present as a source of osteoblasts (Francois and Blanc,

1956 ). If this is true, then the cells of the blastema are not so fully dedifferentiated as their appearances might lead one to believe.

Fin rays also regenerate in the unamputated fin. This can be observed under two conditions. If a hole is cut ih the middle of a fin, or if a length of ray is picked out of an otherwise intact fin, the miss­ ing parts are also regenerated. In the former case, the type of response depends upon the size of the hole. Large holes, several rays across, regenerate by forming a blastema along the proximal edge which grows out in the usual manner. In rare cases. regeneration may also occur from the distal margin, in which case new fin tissue grows proximally. Therefore, it is possible for a fin to grow new parts in opposite directions at the same time, but despite the reversal of ,, polarity such outgrowths are always morphologically distal with re­ spect to the cut surfaces from which they are produeed (Goss. 1956 ).

In the case of smaller holes, the healing process usually fills in the aperture before ray regeneration gets underway. When it finally occurs. replacement extends in the distal direction from the proximal stump, eventually to meet the other end of the ray before 67

proximally directed regeneration from the latter begins. Sometimes

growth proceeds in both directions, but always sooner and faster from

the basal end. The same thing happens when only a segment of half

a ray are removed instead of the full thickness of the fin. Sometimes,

however, regenerating rays fail to meet up with their more distal

segments. When this happens, they may grow on past into the adjacent

interradial region (Comfort and Doljanski, 1958).

Ray regeneration always occurs in the same way, whether in

the outgrowths from amputated fins, or within the tissues of the fin

following extirpation of parts of rays. New rays invariably develop in

the epidermal basement membrane. Aside from blastema formation,

the only difference to be found between fin regeneration and ray re­

generation, is that actinotrichia are not produced in association with the latter phenomenon. Whatever their role may be, actinotrichia appear not to be necessary for ray regeneration alone, but are some­ how involved in the replacement of the fin as a whole (Birnie, 1934).

Fin regeneration can be investigated not only by means of deletion experiments as above, but also by the opposite technique of putting extra rays into the fin. It is possible, with a little care, to insert a length of bony ray into a subcutaneous funnel made in the interradial region of the fin. When subsequently amputated through such transplants the fin regenerates correspondingly supernumerary rays (Goss and Tassava, 1966). This is compelling evidence that rays induce their own regeneration. Not only that, but they also 68

determine the kind of ray to be produced. For example., in the gold­

fish tail some rays are long and others are short. If long ones are

grafted in between short ones, or vice versa, their regeneration will

be true to type. That is., long rays grow faster and farther than

short rays, no matter where they may be located in the fin.

The extent of fin regeneration, of course., depends upon how

much was cut off in the first place (Goss., 1957; Conant, 1970). Fish

fins grow back to their original dimensions before they stop regenerat­

ing and resume the slower rate of elongation to keep up with normal

body growth. However, the rate at which fins regenerate is a function

of the level of amputation. Early in this century, Thomas Hunt

Morgan showed that if a fin is amputated diagonally, the more proxi­

mally severed rays grow faster than do those amputated distally.

Thus, they all reattain their original lengths at approximately the same

time. A fin, however, does not elongate at the same rate throughout the course of its regeneration. After a slow start, when healing and

blastema formation are going on., the rate of regeneration soon

reaches a maximum and very gradually declines thereafter. Regard­ less of how much of the fin has been amputated, the same pattern of

regeneration is followed on very much the same time scale. The only factor so vary significantly is the magnitude of the growth rate, which turns out to be greater at the more proximal levels of amputation

(Morgan 1901). 69

Why it is that proximal levels of a fin grow faster than distal

ones we do not know. It is not correlated with the cross-sectional

area of the stump~ nor with the percentage of the fin amputated.

Possibly the decreasing growth rate along the length of a fin may be a

function of its innervation or blood supply; or perhaps the explanation

'resides in some subtle quality inherent in the cells at different posi­

tions in the fin. This fascinating problem has thus far eluded all

attempts to fathom the mysteries of how growth rates are regulated.

At each level in a fin the tissues seem to be coded for exactly

what lies distal to them (Tassava & Goss~ 1966; Conant. 1970). Up::>n

amputation this is precisely what they reproduce. Their information

content~ however~ is qualitative as well as quantitative~ for they

always produce the correct kinds of things in just the right locations.

This is most dramatically illustrated in the case of the pigment patterns on fins. If a fin has a black spot on it. for example. which may be com­ pletely removed by amputation~ the regenerating fin will reconstitute a replica of the original spot. In the zebra fish~ Brachydanio~ fin re­ generation occurs in technicolor (Goodrich and Greene. 1959). The anal fin of this fish has red, black. and yellow stripes running horizon­ tally across it at nearly right angles to the fin rays. Yet when these stripes are removed by amputation. new ones differentiate as the fin itself. regenerates. They do so by virtue of the invasion of the regene­ rate by undifferentiated pigment cell precursors. When these cells 70

arrive at their proper locations they then become pigmented accord­

ingly. It is a moot question, however, whether the color of pigmenta­

tion is determined by the location of the cell in the fin, or whether the

distance a cell migrates along the rays is conditioned by the kind of

pigment cell percursor it has already been determined to become.

From all the above experiments no one has reported body re­

generation in Zebrafish, Brachdanio rerio. This thesis is on the posterior body regeneration of Zebrafish.

.•. · MATERIALS AND METHOD

Adult Zebrafish, Brachydanio rerio were kept and fed in our

laboratory. They were fed with conditioning foods, growth foods,

staple foods and live brine shrimps.

Before breeding, the males and the females were separated

for a period of 24 hours to get them sexually excited. The females

used for breeding were selected on the basis of having a heavy belly

suggesting matured eggs. The breeding tank was partitioned with a

net two inches below the water level to prevent the adult fish from

eating up the fertilized eggs (See Figure 1 ). The breeding tank con­ tained three adult Zebrafish (one female and two males). They were

left in this position overnight and breeding took place at sunrise.

Zebrafish is a tropical fish, hence we kept the temperature of the tank at 28° C. Tap water was used after it had been treated with commercial Water Rite to eliminate the chlorine.

The fertilized eggs were removed from the bottom of the tank and kept in a smaller tank where they developed. The temperature of the smaller tank was 26° C. Hatching took place on the fourth day.

The young fish were allowed to develop for 6 days after hatching be­ fore any amputations were performed. Zebrafish are not hardy during .•"' . 5 Jittt

72

these early stages of development. To ensure enough animals for

this study, 180 6 -day fry were amputated and allowed to develop. Of

these 110 survived. The data presented was obtained from these 110

which survived. The young fish were anaesthetized with MS222

(Tricaine methane sulfonate) one part to 2, 000 parts of distilled

. water. Transverse cuts were made on these young fish 2 ocular

micrometer units posterior to the Cloaca. The amputated fish were

cultured in finger bowls containing 200 C. C. of dechlorinated water.

Ten young fish were placed in each fingerbowl. Five young fish were

used for studies each day from the day of amputation to the 8th day

when there was no more blastema to be measured. The remaining

70 fish were observed to the 30th day when regeneration was completed.

These fish were killed and fixed in Tellysnicky Fluid (a mixture of

Bichromate of Potassium and acetic acid. A solution of Bichromate

of Potassium was made by dissolving 6 grams of it in 200 cc of water.

The glacial acetic acid was made by mixing lOcc of the acid to 200cc

of water. 200cc of the Bichromate solution with lee of the acid was

used as the fixative).

After fixation, the fish were photographed, for the gross mor­

phological studies. The length of the regenerated portion was

measured from the point of amputation and the average of the previous

days' growth was subtracted to get the current daily growth. In addi­

tion description of the developmental stages were given. L 73

Mortality rate was quite high during the early stages. These fish are extremely susceptible to environmental factors. The labora­ tory temperature also fluctuated drastically. Also bacteria, fungus and protozoa might have invaded the cut surfaces.

These two possible causes of mortality were immediately taken care of in the following ways: the temperature of the water was kept constant by the use of Thermostatically controlled heat. Antibiotics

(a mixture of Streptomycin and Penicillin) solution were added to the water. The Streptomycin and the Penicillin were mixed in a 1:1 ratio; 0. 01:0. 01 in a liter of water. EXPERIMENTAL RESULTS

A rather clear pattern of regeneration was observed and, although there were small individual variabilities (depending on the size and the angle of cut) the growth forms may be generalized for descriptive purposes. Great care was taken in cutting because tails cut on a bias are characterized by angular growth. (See figures 2., 3, and 4).

The tail of Zebrafish demonstrates a notable capacity for

Self-replacement during the first month of its life. The initial direc­ tion of regenerative growth depends upon the angle of the amputation plane. If the cut is at an an angle, rather than transverse, the axis of the regenerate is at right angle to the amputation plane.

Growth curve showed a lag period the first day of amputation, a period of rapid growth lasting 3 days .. and then a time of decelerat­ ing growth. Regeneration was completed on the 30th day following amputation. Cutting usually elicits very little bleeding and is followed immediately by contracture. Wound healing takes place within a day (epidermis migrate over the cut surface).

Figure 5 shows the appearance of the fish on the day of ampu­ tation. The notochord, muscle, dorsal and ventral fins, blood vessels,

74 75

spinal cord have been sever

fish oneday'after amputation.

The wound is completely healed. There is a terminal swell­

ing at the cut surface which has all the gross signs of a Blastema.

Grossly there is no visible observable changes between that of

the first day and those of second and third day except that there is an

increase in the size of the blastema.

Figure 7 shows the appearance of the fish on the fourth day.

The melanophores are new apparent.

The ventral and dorsal fins which were also amputated along with the tail continue to grow from the cut surface till they join to form the caudal fin (See Figure 14). Figure 8 is the appearance of the fish 5 days after amputation and. Figure 9, six days after amputa­ tion. By the eighth day fin rays become apparent (See Figure 10).

At this stage the tail fin is diphycercal type which is typical of primi­ tive fish (Morgan. 1902 ). This supports the theory that "Ontogeny re­ capitulates phylogeny. " An adult zebrafish has a homocercal caudal fin (ray fins); embryonic animals behave like their ancestors. Figure

11 shows the regenerate on the 12th day and Figure 12 shows the appear­ ance of the regenerate on the 18th day.

Regeneration was completed on the 30th day (Figure 13). The caudal fin assumes a familiar homocercal pattern typical of teleosts

(Goss and Stagg. 1957; Bernie, 1934). DISCUSSION

This work has investigated the regenerative capacity of teleost fish typified by the Zebrafish, Brachydanio rerio. Spallanzani in

1786 suggested that teleosts might have the ability to regenerate lost tail. His postulate has been proven to be correct by Conant, 1970, who worked with African lung fish, Protopterus, Goss · 19 56, and by the present work.

It is consistent with the evolutionary position of the Teleosts that regeneration in Brachydanio rerio bears resemblance to both other fish and tetrapods: like Osteichthyes in overall regenerative capacity, like tetrapods in growth form. Of all vertebrates amphi­ bians and fish possess the greatest capacity for regeneration: limbs in amphibians (Barber, 1944; Manner, 1953; Glade, 1963; Dont, 1962;

Haas, 1962) and in fish, tail (Conant, 1970; Kamrin, 1955;) and fins,

(Nicholas, 1955; Goss, 1956). Scales and barbels all have great capa­ city for regeneration, (Goss, 1954; Kamrin and Singer, 1955). What general concepts can be derived from this survey of Regeneration?

The ability to regenerate is widespread among animals. In many this ability is enormous, whereas in others it is very limited or even lacking. It brings into proper perspective a point which has been

76 77 emphasized by students of regeneration since the early days~ i.e.~ that there is a correlation between regeneraion potential and level of organisation. Lower organisms have a high regenerative power~ and this power decreases with increase in complexity, (Hamburger~ 1965;

Morgan, 1901; Korschelt, 1927; Broussonet, 1786).

Mammals, including man, pay for their high organisation with an almost complete loss of regenerative capacity. The relation be­ tween regenerative power and level of organization, however, holds only in a general way and breaks down in numerous special instances.

For example, some planarians have an exceptionally high regenerative power, but closely related species have none (Hamburger, 1965). The same holds /for Annelida (Morgan, 1901). The regeneration of the crystalline lens of the eye 'Of salamanders is perfect in one genus and absent in another. These examples could be multiplied easily. They show that factors other than the general level of organisation of the animal often play a decisive role.

It has also been asserted that since regeneration is a develop­ mental process, embryos, larva and young animals, in general, should be more readily capable of regeneration than adult stages of the same animal. This expectation is fulfilled in many instances but again is not a rigid rule. It is true that Salamander larvae regenerate limbs and tails more readily and more perfectly than adults do, and lens re­ generation in some Salamander species is limited to larvae. Frog 78 tadpoles can regenerate a hindlimb but the adult frog cannot (Goss.

1956). On the other hand sea urchin and starfish larvae do not re­ generate. while adults do (Hamburger. 196~). The same holds good for annelids and for tunicates.

Again. factors other than age determine the limitation of re­ generative power in these forms. Finally. one would expect that external organs and appendages which are readily exposed to injury and loss would show a higher regenerative capacity than inner organs.

This again, holds in a general way. However. several examples have been mentioned of the regeneration of inner organs which are rarely subject to injury during the normal course of life.

Brachydanio rerio fits into the pattern of regeneration seen in lungfish. toads and salamanders. This work has shown that Zebrafish do indeed regenerate.

The next step is to determine the histological changes and patterns occurring during the 30 day period of regeneration in Zebra­ fish. Does each of the tissues of the stump give rise to newly regene­ rating tissues on a one to one basis. notochord to notochord. muscle to muscle. connective tissue to connective~ etc. etc. ? In what time order do these occur? Do muscles form first. then notochord. then blood vessels. then nerves? In what order do they occur? These are the questions that still need to be answered.

We now know that. at 10 days old, Zebrafish have the capacity 79 to regenerate. From my work and from the work done by others

(Conant, 1970), it seems that the more primitive a fish is, the more regenerative ability it has. CONCLUSION

The regeneration of the posterior body region of Zebrafish.

Brachydanio rerio has been studied. A 10 day old Zebrafish demon-

. strates a notable capacity for self replacement. The initial direction

of growth in the regenerate depended upon the nature of the amputation

plane: straight. pointed, or terminally notched cuts elicited new

growth along the primary axis. whereas cuts made at an angle re­

sulted in regenerates whose axis was perpendicular to the plane of

cut.

Growth curve showed a lag period. a period of rapid growth

and then a time of decelerating growth.

Tails cut proximally showed greater and more rapid growth

than those with less material removed.

The present work has shown that young Zebrafish have the

capacity to regenerate their posterior body region.

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APPROVAL SHEET

The thesis submitted by Gabriel J. Ekandem has been read

and approved by the director of the thesis.

Furthermore, the final copies have been examined by the

. director and the signature which appears below verifies the fact

that any necessary changes have been incorporated, and that the

thesis is now given final approval.

The thesis is therefore accepted in partial fulfillment of

the requirements for the degree of MASTER OF SCIENCE.

Date

l