Book - The Frog Its Reproduction and Development 1

Chapter 1 - Introduction

The Period of Descriptive Embryology Epigenesis vs. Preformationism

The Period of Comparative Embryology Soma and Germ Plasm

The Period of Cellular Embryology The Germ Layer Concept

The Period of Experimental Embryology The Normal Sequence of Events in

The Embryologist as a Scientist Embryology

Why the Embryology of the Frog? Cell Multiplication

Some General Concepts in Embryology Cell Differentiation (Specialization)

Biogenesis Organogeny

Biogenetic Law — The Law of Re- Growth capitulation

Embryology is a study of early development from the fertilized egg to the appearance of a definitive organism. The stage is generally referred to as its embryonic stage. It is not so inclusive a term as ontogeny, which refers to the entire life history of an organism from the fertilized egg to old age and death. Since either the sperm or the egg (of a zygote) can affect development, the study of embryology might well begin with a study of the normal production and maturation of the germ cells (gametes). It should include fertilization, cleavage, blastula and gastrula formation, histogenesis, organogenesis, and the nervous and humoral integrations of these newly developed organs into a harmoniously functioning organism. When development has proceeded to the stage where the embryo can be recognized as an organism, structurally similar to its parents, it no longer can be regarded as an embryo.

The unfertilized egg is an organism with unexpressed potentialities derived from an ovary containing many essentially similar eggs. Its basic pattern of development is maternally derived and is predetermined in the ovary but its genetic complex is determined at the very instant of fertilization. This predetermination is in no sense structural (i.e., one never has seen a tadpole in a frog's egg) but is nonetheless fixed by both the nuclear and the cytoplasmic influences of the fertilized egg (or zygote). These influences are probably both physical and chemical in nature.

No amount of environmental change can so alter the development of a Starfish zygote (i.e., fertilized egg) that it becomes anything but a starfish, or change the potentiaHties of a fertilized frog egg into anything but a frog. This means that the nucleus and the cytoplasm of the fertilized egg together possess certain potentialities, as well as certain limitations, in development. Within the range of those limitations the potentialities will inevitably become expressed, in any reasonably normal environment.

It is now quite clear that both the nucleus and the cytoplasm are influential in development. The genetic influences are largely nuclear, but cellular differentiation is cytoplasmic. With each of the cleavages there is a synthesis of nuclear material (e.g., desoxyribose nucleic acid) out of the cytoplasmic (ribonucleic acid), and cytoplasmic from the yolk or other extrinsic food sources. Development is not simply growth or increase in mass. It involves a constant synthesis or the building-up of those elements so vital to the normal processes in the development of the individual. Each stage is built upon the successful completion of the preceding stage.

Frog's egg and swollen jelly shortly after fertilization.

Comparative embryologists have found that there is a somewhat similar pattern to the development of all forms. The earlier in development that the comparison is made, the greater the similarity among even widespread species. This may mean that all other possible types of development have failed to survive in a harshly selective environment. Or it may suggest a common ancestry. In any case, all embryos do begin with the fertilized egg. (With the rare exceptions of natural parthenogenesis.) The activated egg immediately manifests certain metabolic changes which may be correlated with the kinetic movements leading toward the completion of maturation or toward the first cleavage. Division results in a shifting of the nucleo-cytoplasmic volume relations from the unbalanced condition of the ovum to the more stable ratio found in the somatic cells. Each division of the egg means more nuclear material, less cytoplasmic material, and more rigid cells. This rigidity, the adhesiveness of cells, and their activity together cause the development of sheets of cells which soon cover (chick) or surround (frog) a cavity, known as the blastocoel. As this sheet of cells expands it becomes necessary for it to fold under (chick), to push inward (frog), or to split into layers (mammal), and thereby form the 2-layered gastrula. In most forms almost immediately the third layer (mesoderm) develops between the two preceding layers, epiblast and endoderm. After the derivation of the mesoderm from the outer layer, the latter is then known as the ectoderm. All of this occurs before the appearance of any discernible organs. There is reason to believe that these sheets of cells have topographical rather than functional significance and that parts of any one of them could be exchanged experimentally with any other at this time without seriously disrupting the normal development of the embryo.

At this point, however, the process of cell division becomes secondary to the process known as differentiation or specialization of cell areas. This is the very beginning of organ formation. Many embryos begin to develop transient (larval) organs which are replaced as the more permanent organs appear and begin to function. Eventually there is produced a highly complex but completely integrated organism which is able to ingest, digest, and assimilate food, and subsist for itself independently of its parent organism. It is then no longer considered an embryo.

This period of ontogeny is the subject matter of embryology. We attempt not to answer the major biological question "why" but rather to confine ourselves to a detailed description as to "how" an organism is produced from a fertilized egg. It is the purpose of this book to describe "how" the frog develops from the fertilized frog's egg, as we understand it. The facts of this book are made available through the accumulated studies of many investigators, in this and other countries.

There are those who are interested in the subject matter of embryology solely because they were once embryos themselves, or because they anticipate becoming partners in the further production of embryos. The closer we can come to the understanding of the mechanism of embryonic development of any single species, the closer will we come to the understanding of the mechanism of life itself. The processes of the embryo are certainly fundamental to all of life. Life exists by virtue of successful embryonic development. There are few things in the living world more absorbing or more challenging to watch than the transformation of a single cell into a complex organism with its many organs of varied functions, all most efficiently integrated.

Embryology is not a new subject. It has a very rich heritage, based upon the solid foundation of pure descriptive morphology. This is followed by the comparison of the variations in development, leading to the recent trend toward the physical and chemical analyses of the developmental processes. In order that we do not lose sight of this heritage, a brief survey is given in the following pages.

The Period of Descriptive Embryology

While organisms must have been reproducing and developing since the beginning of life, knowledge of these processes seems to have begun with Aristotle (384-322 B.C.), who first described the development and reproduction of many kinds of organisms in his "De Generatione Animalium." Since he could not locate the small mammalian egg, he considered its development to be the most advanced of all animals. Below the mammal he placed the shark, whose young develop within the body of the female but are born alive and often with the yolk sac attached. Next, below the sharks, he placed the reptiles and the birds whose eggs are complete in that they are provided with albumen and a shell. The lowest category of development was that of the amphibia and fish, which had what he termed "incomplete" eggs, referring, no doubt, to the method of cleavage. Aristotle believed that development always proceeds from a simple and formless beginning to a complex organization characteristic of the adult. Basing this observation on his study of the hen's egg, he laid the foundation for the modern concept of epigenesis or unfolding development. This concept is opposed to the idea of structural preformation of the embryo within the gamete, or germ cell.

William Harvey (1578-1657) long ago came to the conviction that all animals arise from eggs: "Ex ovo omnis"; and later Virchow (1821-1902) went even further to state that all cells are derived from preexisting cells: "Omnis cellula e cellula." Flemming stated: "Omne vivum ex nucleo" and later Huxley said, "Omne vivum ex vivo." These concepts, taken for granted today, are basic in biology and emphasize the fact that only through the process of reproduction has the present population of organisms come into existence. Embryonic development is a prerequisite to survival of the species. Therefore, only those organisms that survive their embryonic development and achieve the stage of reproductive ability can carry the baton of protoplasm from the previous to the future generations in the relay race of life with time.

Fabricius (1537-1619) and Harvey were both limited in their studies by the lack of the miscroscope but both of them presented remarkable studies of early chick development. Malpighi (1628-94), using the newly invented microscope, gave us two works in embryology: "De Formatione Pulli in Ovo" and "De Ovo Incubato," which deal largely with the development of the chick from 24 hours to hatching. Beginning at this stage he was misled to believe that all the various parts of the embryo are preformed within the egg (since chick embryos incubated for 24 hours exhibit most of the major organ systems) and that the process of development was one simply of growth and enlargement. This was the theory of "preformationism" which is diametrically opposed to that of epigenesis or unfolding development. This new concept of preformationism found a staunch supporter in Swammerdam (1637-80). In 1675 Leeuwenhoek firmly believed he discovered the human form sitting in a cramped position in the head of the human spermatozoon. This led to the "spermist" school of preformationists.

It was inevitable that, when parthenogenesis was discovered, the spermists would have to retire in favor of the ovists (e.g.. Bonnet) since eggs were known to develop into organisms without the aid of spermatozoa. This led to the equally ridiculous concept of the "emboitement" or "encasement" theory, which suggested that each egg contains, in miniature, all the future generations to be developed therefrom. Each generation was achieved by shedding the outermost layer, like Chinese ivory boxes carved one within another. But pre formationism of either school was destined to lose adherents as the microscope became further refined. Caspar Friedrich Wolff (1733-94) attacked the idea of preformationism and supported epigenesis on purely logical grounds, put forth in his "Theoria Generationis." In 1786 he published the most outstanding work in the field of embryology prior to the works of von Baer. It was entitled "De Formatione Intestinorum" and in this treatise Wolff showed that the intestine of the chick was developed de novo (epigenetically) out of unformed materials.

The Period of Comparative Embryology

Up to about 1768 embryology was almost exclusively descriptive and morphological. It was inevitable that the second phase in the history of embryology would soon develop, and would be of a comparSpermist's conception of the ative nature. This approach was stimuhuman figure in miniature j^^^^ ^ q^^-^^^ (1769-1832) and his within the human sperm. (Re- , . .• . t

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Hartsoeker: 1694.) 1824 Prevost and Dumas first saw cleav age or segmentation of an egg in reptiles. In 1828 von Baer published his "Entwicklungsgeschichte der Tier" and thereby became the founder of embryology as a science. He established the germ layer doctrine, proposed a theory of recapitulation, and made embryology truly comparative. From a study of the development of various animals, von Baer arrived at four important conclusions, which are known collectively as the laws of von Baer. They are as follows:

1. "The more general characteristics of any large group of animals appear in the embryo earlier than the more special characteristics."

2. "After the more general characteristics those that are less general arise and so on until the most special characteristics appear."

3. "The embryo of any particular kind of animal grows more unlike the forms of other species instead of passing through them."

4. "The embryo of a higher species may resemble the embryo of a lower species but never the adult form of that species." (This latter statement is the basis of the Biogenetic Law when it is properly interpreted.)

Following von Baer, Kowalevsky (1866) stated that all animals pass through a gastrula stage. Haeckel (1874) proposed a Gastrea Theory which suggested that the permanent gastrula, the adult Coelenterata, might be the form from which all higher diploblastic (gastrula) stages in their life history were derived.

The Period of Cellular Embryology

As the microscope was still further refined, and embryos could be studied in greater detail, it was natural that a further subdivision of the field of embryology would occur. In 1831 Robert Brown discovered the nucleus; in 1838 Schleiden and Schwann founded the cell theory; in 1841 Remak and Kolliker described cell division; in 1851 Newport observed the entrance of the spermatozoon into the frog's egg; and in 1858 Virchow published his "Cellular Pathology." Later, in 1878, Whitman and Mark initiated the study of cell lineage ("Maturation, Fecundation, and Segmentation of Limax") by which the fate of certain early blastomeres of the embryo was traced from their beginning. In 1882 Flemming discovered the longitudinal splitting of chromosomes; and Sutton in 1901 gave us the basis for the modern chromosomal theory of inheritance. The study of the embryo was thus broken down into a study of its constituent cells; then to the nucleus; and finally to the gene. From the morphological, physiological, or genetic aspects, this field of cellular embryology is still very active.

The Period of Experimental Embryology

During the latter part of the nineteenth century, investigators began to alter the environment of embryos, and surgically and mechanically to interfere with blastomeres and other parts of the developing embryo. This new "experimental" approach required a prior knowledge of morphological and comparative embryology. It was hoped that, by altering the physical and chemical conditions relating to the embryo, the normal mechanism of development would be understood better through an analysis of the embryonic adjustments to these alterations. Before the turn of the century, the earlier workers in this field were His, Roux, Weismann, Born, Driesch, and the Hertwigs (Oscar, Richard, and Paula). Then came Morgan, Spemann, and Jacques Loeb. Finally during the last several decades there has developed a host of experimental embryologists, many of whom were inspired by association with the above workers. Reference should be made briefly to Adelmann, Baltzer, Bataillon, Bautzmann, Boell, Brachet, Child, Conklin, Copenhaver, Dalcq, de Beer, Detwiler, Ekman, Fankhauser, Goerttler, Guyenot, Hadorn, Hamburger, Harrison, Herbst, Holtfreter, Just, Korschelt, Lehmann, the Lewises, the Lillies, Mangold, Nicholas, Oppenheimer, Parmenter, Pasteels, Patten, Penners, Rawles, Rotmann, Rudnick, Schleip, Schultz, Spratt, Swingle, Twitty, Vogt, Weiss, Willier, E. B. Wilson, and a host of others.

A further refinement of this approach is in the direction of chemical embryology or a study of the chemistry of the developing embryo and the raw materials from which it is formed. As Needham ( 1942) says: "Today the interest has been shifted to the analysis of the fundamental morphogenetic stimuli which operate in embryonic life." Such stimuli as these may well be of an ultra-chemical or ultra-physical nature.

Embryology as a division of science has gone through a period of its own development from the purely descriptive phase to the presentday biochemical and biophysical analysis of development. However, each generation must recapitulate this sequence; therefore each student must begin with the foundation of basic, morphological embryology before he can expect to comprehend the possibilities in the superstructure of the experimental approach.

It is the function of this particular book to provide the student with the foundational information relative to one genus, namely the common frog. This will introduce him to the major aspects of embryonic development and at the same time give him a factual foundation upon which he may later make his contribution in one of the fields of embryology. Where it will not confuse, but might clarify the developmental process, reference will be made to specific findings in the field of experimental embryology.

The Embryologist as a Scientist

There are certain characteristics necessary for success and satisfaction in pursuing the study of embryology, or, in fact, any science. Some of these may be inherent, but it is more reaHstic to assume that they can be developed.

First: One must have the completely open mind characteristic of any true scientist. A student must come to any science without bias, without preconceived or prejudiced concepts. He must be willing to say: "Show me, give me proof, and then Fll believe." Science is a body of knowledge, accumulated through generations by fallible human beings. It is therefore as reliable as human experience but it is subject to change with knowledge gained through further human experience. Basically this body of demonstrated fact can be accepted at its face value as a foundation upon which to build. It is a heritage of generations of trial and error, of observation, experimentation, and verification. But the scientific attitude presumes that there is still much to learn, and some ideas to be revised. The scientist maintains an open mind, eager to be shown and willing to accept demonstrable fact.

Second: The student of embryology must have the ability to visualize dynamic changes in a three-dimensional field. Most embryos are not naturally transparent and it is difficult to observe directly what is

Posterior

FRONTAL SECTION -LATERAL VIEW

FRONTAL SECTION- DORSAL VIEW Dorsal

TRANSVERSE SERIAL SECTIONS - LATERAL VIEW SAGITTAL SECTION - LATERAL VIEW

Planes in which the embryo may be cut or sectioned.

transpiring within. It is necessary, therefore, to study such embryos after they have been preserved, sectioned, and stained. Nevertheless, the emphasis in embryology is on the dynamic changes that occur, on derivations, and on the end organs of the developmental processes. One is not interested in a single frame of a moving picture nor is it sufficient to describe all of the structures in a single section of an embryo. The student of embryology is interested in the composite picture presented by a succession of individual frames (sections) which are re-assembled in his mind into a composite, three-dimensional whole. It is necessary, in dynamic embryology, to reconstruct in the mind the inner processes of development which proceed from the single-celled egg to the multicellular organism functioning as a whole. The embryo also must be regarded in the light of its future potentialities. While parts of the embryo are isolated for detailed study, the student must of necessity re-assemble those parts into a constantly changing threedimensional whole. The embryo is not static in any sense — it is dynamic.

Third: The student of embryology must have an intelligently directed imagination, one based upon a foundation of scientific knowledge. He must be rigidly loyal to demonstrable fact, but his mind must project him beyond those facts. It is through men with intelligent imagination that there have been such remarkable advances in the superstructure of embryology. Coupled with a healthy curiosity, such a characteristic is causing men constantly to add facts, which withstand critical investigation, to our ever-increasing body of knowledge.

Why the Embryology of the Frog?

It is the contention of the author that the student who understands thoroughly the development of one species will have the foundation for the understanding of the basic embryology of all species. This does not imply similarity in development to the extent that there is no room for comparative embryology of such forms as Amphioxus, fish, frog, reptile, bird, and mammal. But the method of study, the language used, and the fundamental processes are sufficiently alike so that a thorough understanding of one form will aid materially in the understanding of the embryology of any other form. It is too much to catapult a student into the midst of comparative or experimental embryology and expect him to acquire any coherent conception of normal development.

The normal embryology of the frog is well understood, and there is no better form through which to introduce the student to the science of embryology. The frog is a representative vertebrate, having both an aquatic and a terrestrial existence during its development. This metamorphosis requires, for instance, several major changes in the respiratory and excretory systems during development. The embryology of the frog illustrates the sequence of developmental events (organogeny) of all higher vertebrates. Finally, the living embryos of the frog are now available at any season of the year and students can observe directly the day-to-day changes that occur from fertilization to metamorphosis.

Some General Concepts in Embryology

Biogenesis

There is no substantiated evidence of spontaneous generation of life, although, as a scientist, one cannot deny its possibility. All life does come from preexisting life. In embryology we are more specific and state that all organisms come from eggs. This statement is certainly true of all Vertebrates and of the vast majority of animals. However, it cannot be said of the single-celled Protozoa or of some of the lower Invertebrates which reproduce by binary fission, spore formation, or budding.

All protoplasm in existence today is believed to be descended from preexisting protoplasm and it is therefore related by a continuous line from the original protoplasmic mass, whenever that came into existence. Further, sexual organisms living today are each descendants of a continuous line of ancestors not one of which failed to reach the period of sexual productivity. So, the mere existence of an organism today is testimony of its basic relationship to all protoplasm and to its inherited tendency toward relative longevity. At present one cannot conceive of life originating in any manner but from life itself. All theories are, of necessity, philosophic speculation.

Biogenetic Law — The Law of Recapitulation

The original laws of von Baer are very clearly stated, but Haeckel and others have misinterpreted or elaborated on them so that a confusion about these concepts has arisen in the popular mind. Von Baer clearly emphasized the fundamental similarity of certain early stages of embryos of various forms. He never suggested that embryos of higher forms recapitulated the adult stages of their ancestors; that man as an embryo went through such stages as those of the adult fish, amphibian, reptile, bird, and finally anthropoids and man. The similarities referred to were in the early developmental stages, and most definitely not in the adults.

As one studies comparative embryology it becomes clear that there is a basic similarity in development, and that the brain, nerves, aortic arches, and metameric kidney units (for instance) develop in a somewhat similar manner from the fish to man. Proponents of the theory of evolution have emphasized this as evidence in support of their theory. It is, however, possible that this similarity is due to a highly selective environment which has eliminated those types of development which have digressed from a certain basic pattern. Put another way, the type of development we now know was suited to survive in the environments available. Three primary germ layers may have proved to be more efficient (i.e., to have better survival value) than two or four, and so the gradual transition from pro- through mesoand finally to the meta-nephros may be a necessary corollary of the slow developmental process. Von Baer's laws may simply represent one method of expressing a fundamental law of nature (i.e., survival values) rather than a phylogenetic relationship.

There are many specific instances in development which could be used to refute the Law of Recapitulation as it is often stated, but which would in no way refute von Baer's "Biogenetic Law." One example may be cited: there is no evidence among the lower forms of any anticipation of the development of the amnion and the chorion of the amniotes. The Biogenetic Law was derived from a study of comparative embryology, and possibly it has significance in the understanding of the mechanism of evolution.

Epigenesis vs. Preformationism

The historical sequence from preformationists to spermists and then ovists was very natural. The refinement of optical equipment dispelled these earlier concepts and the pendulum swung to the opposite extreme where embryologists believed that nothing was preformed and that development was entirely epigenetic. As often occurs, the pendulum has swung back to an intermediate position today.

No one has ever seen any preformed structure of the organism in any germ cell. Nevertheless, one can bring together in the same receptacle the fertilized eggs of closely and distantly related forms and yet their individual development is unaffected. A normally fertilized egg of the starfish will always develop into a starfish, a frog's egg into a frog, a guinea pig egg into a guinea pig. The environment of the fertilized egg has never been known to cause the transmutation of species. Further, we cannot see the stripes on a 2-cell mackerel egg, or the green and yellow spots on the 2-cell frog's egg, or the colorful plumage on a peacock blastoderm, or the brown eyes of the human optic vesicle. Nevertheless these intrinsic genetic potentialities are definitely preformed and inevitable in development under normal environmental circumstances. Development is epigenetic to the extent that one cannot see the formed structures within the egg, or the sperm, or zygote. However, the organism is preformed chemically (genetically) to the extent that its specific type of development is inevitable under a given set of circumstances. Our modern interpretation is therefore intermediate between that of preformationism and epigenesis. There is unfolding development within certain preformed limits which must be chemical and/or physical, but not visibly morphological.

Soma and Germ Plasm

It is definite that in some forms there is an early segregation of embryonic cells so that some will give rise to somatic (body) tissues while others will give rise to germ (reproductive) tissues. This was the contention of Weismann and many others about 1900. The germinal cells seem to come from the extra-embryonic regions of many vertebrates and become differentiated at an early stage of development. Whether or not these migrating pre-germ cells are the precursors of the functional gametes has not been determined conclusively. Nevertheless, the germ plasm, once segregated, is immune to damage by the somatoplasm. This has been illustrated many times by men who have incurred injuries and yet who have subsequently produced normal offspring. The germ plasm is therefore functionally isolated, segregated in the adult. Further, x-ray damage to the germ plasm does not show up in the somatoplasm at least until the next generation.

Germ plasm can and does give rise to somatoplasm during normal embryonic development. There is little, if any, evidence that somatoplasm, once formed, can give rise to germ plasm. When the fully formed gonads of higher animals are removed in their entirety, there seems to be no regeneration of germinal tissues from the remaining somatic tissues.

However, modern embryologists are rather reluctant to believe that these two types of protoplasm are as fundamentally segregated from each other as was once believed. Regeneration is a characteristic of lower forms. In these cases it certainly involves a redevelopment of germinal tissue (e.g., Planaria regeneration of a total organism from one-sixth part completely devoid of germinal tissue). The lack of regenerative powers in general among the higher forms may be the intervening factor in the distinction between somatoplasm and germ plasm, and the lack of interchange or regeneration between them.

The Germ Layer Concept

In 1817 Pander first identified the three primary germ layers in the chick embryo, and since then all metazoa above the Coelenterata have been proved to be triploblastic, or tri-dermic. The order of development is always ectoderm, endoderm, and then mesoderm, so that the most advanced forms in the phylogenetic scale are those possessing mesoderm.

We tend to forget that these germ layer distinctions are for human convenience and that the morphogenetic potentialities are relatively unaware of such distinctions. One can exchange presumptive regions of the blastula so that areas normally destined to become mesoderm may remain in a superficial position to function as ectoderm, or become endoderm. The exchanges may be made in any direction in the early stages. The embryo as a whole may develop perfectly normally, with exchanged presumptive germ layer areas.

When we remember that all tissues arise from cells having the same origin (i.e., from the zygote), and that mitosis ensures similar qualitative and quantitative inheritance, then the presumed distinctions of the three primary germ layers seem to dissolve. It is only during the later phases of development (i.e., during differentiation) that the totipotent genetic capabilities of the cells become delimited, and we have the appearance of structurally and functionally different cell and tissue types.

The Normal Sequence of Events in Embryology

Cell Multiplication.

From the single cell (the fertilized egg or zygote) are derived the many millions of cells which comprise the organism. This is done by the process of almost continual mitosis or cell division involving synthesis and multiplication of nuclear materials. This continues throughout the life of the organism but it is at its greatest rate during embryonic development.

Cell Differentiation (Specialization).

After gastrulation the various cell areas, under new morphogenetic influences, continue to produce more cells. However, some of these cells begin to lose some of their potentialities and then to express certain specific characteristics so that they come to be recognized as cells or tissues of certain types. Generally, after this process of differentiation has occurred, there is never any reversion to the primitive or embryonic condition. Differentiation is cytoplasmic and generally irreversible.

Organogeny.

The formative tissues become organized into organ systems which acquire specific functions. The embryo then begins to depend upon these various newly formed organs for certain functions which are increasingly important for the maintenance and integration of the organism as a whole.

Growth.

This phenomenon involves the ability to take in water and food and to increase the total mass through the synthesis of protoplasm. Growth may appear to be quite uniform in the beginning. However, as the organs begin to develop there is a mosaic of growth activity so that various organ systems show peaks of growth activity at different stages in embryonic development. It may be said that embryonic development is completed when differentiation and organogeny have been fully achieved.