In this article we will discuss about the analysis of nature and action of organizer in amphibian eggs.
Grey crescent—its Role in Development:
Wilhelm Roux (1888) first described the grey crescent in amphibian egg. The experiments of Rund and Spemann have emphasised the vital role of grey crescent in amphibian development (see Fig. 5.14). If the egg is surgically divided into two halves, each having one half of the grey crescent, each half of the egg will develop into an entire embryo if cultured in isolation.
The same operation if performed on an egg in which one half gets the entire grey crescent, while the other blastomeres lack the grey crescent material and the two blastomeres when cultured in isolation, the blastomere which is devoid of grey crescent will develop a simple sac of ectoderm containing endoderm, while the blastomere having the grey crescent will form an entire embryo.
Similar operation of removal of grey crescent from the egg prior to cleavage has shown that the process of cleavage remains unaltered, but gastrulation does not occur.
The vital dye method and other marking devices, such as carbon particle technique and radioactive labelling method have been applied to ascertain the fate of grey crescent during development. These experimental studies have revealed that the grey crescent material of the egg gives rise to the dorsal lip of the blastopore in early gastrula.
But in the late stage, i.e., when the gastrula transforms into an embryo, the grey crescent materials become localised in the head endoderm of the primitive gut.
Dorsal Lip of Blastopore and its Significance:
The ectoderm in early gastrula remains in an undifferentiated stage, i.e., it has manifold potencies at that stage. During gastrulation, the potencies of ectoderm become limited from general to specific one. In normal amphibian development the dorsal ectoderm just above the dorsal lip of the blastopore becomes the neural structure.
The dorsal lip region is called the chorda-mesoderm because it develops normally into mesoderm and notochord. Spemann and his student, Mangold, by using a transplantation technique, have shown that the dorsal lip of the blastopore, when implanted to the ectodermal area of the same or another embryo at that stage of development, the dorsal lip is able to initiate the formation of a secondary embryonic axis.
Fig. 5.22 demonstrates the formation of a secondary head as a result of grafting the dorsal lip of the blastopore. They used the dorsal lip of the blastopore of Triton cristatus (Nonpigmented) as the graft to an embryo of Triton taeniatus (Pigmented) of the same age.
This experiment demonstrates that the neural tissue of the secondary embryo is entirely formed from the tissues of the host—thus suggesting the fact that the dorsal lip of the blastopore is not only the controller of development but also acts as an instigator to induce the host tissue to differentiate.
Concept of Embryonic Induction:
The foregoing discussion suggests that the commitment of a particular tissue to a particular developmental fate depends generally on the relationships established with surrounding tissues. The development of amphibian neural plate has shown that the dorsal lip of the blastopore invaginates inside the embryo and induces the dorsal ectodermal layer to form the neural structure.
If the invagination is prevented by treatment of the amphibian blastula with a solution of 0.35% sodium chloride solution as shown by Holtfreter in Exogastrulation Experiment, the dorsal ectoderm that normally becomes neural tube forms the ectodermal vesicle.
So the determination of neural tube is dependent upon the association with the adjoining tissue, such a phenomenon is often referred as embryonic induction.
The transplantation experiment of Spemann and Mangold (1924) with the dorsal lip of the blastopore and the exogastrulation experiment of Holtfreter (1933) with amphibian embryo also suggest another phenomenon in embryonic differentiation.
When the differentiation of a group of cells depends upon another group of cells —such a process is called the dependent differentiation. The development of neural tube is a case of dependent differentiation. When a cell group is capable of differentiation without the intervention of other, cells—the differentiation is called the independent differentiation.
Embryonic Induction:
Embryonic induction is a process whereby a cell group causes another group of cells to differentiate to a particular developmental fate. Such a phenomenon is a recurring process in embryonic development, i.e., there are hierarchies of induction.
Induction of neural tube by the dorsal lip of blastopore material is the first inductive phenomenon in amphibian ontogenesis. So the inductor of the brain development is referred as primary inductor.
Besides this primary one, there are secondary, tertiary inductors, and so on, in hierarchical array. The brain having been induced, develops regional specificity under the influence of head endoderm and mesoderm. The hindbrain exerts inductive influence over the overlying ectoderm to form ear placode which becomes invaginated to form auditory vesicle.
The auditory vesicles, in turn, induce the surrounding mesenchyme to develop into the auditory capsule. The development of eye relates a similar story. The diencephalon forms paired optic vesicles which make contact with the overlying head ectoderm. The ectoderm forms a lens which invaginates to form lens vesicle. The eye-cup-lens complex induces the formation of cornea and associated mesodermal derivatives.
Nature of Induction:
The neural differentiation in amphibian development has shown that the archenteron roof induces the overlying ectoderm to develop into neural structures. This fact suggests a direct influence upon the ectodermal cells by the archenteron roof.
Two alternate possibilities can be suggested:
(1) Surface interaction or
(2) Chemical intervention.
Most of the experimental evidences on this line suggest the existence of chemical mediation between the inductor and induced structures.
Many chemical substances suspended in egg albumen (an inert substance) can induce neural tissue when introduced into the blastocoel of early gastrula. As the implanted material contains no cells—the question of surface interaction of cells at the inductive interface becomes impossible.
Besides, if a portion of presumptive embryonic ectoderm and a piece of dorsal lip of blastopore are kept separated in a culture medium by a millipore filter paper of 20 micra thick with an average pore size of 8 micra, the ectoderm develops into a neural structure. The filter paper prevents the transfer of cells but permits diffusible chemical substances of low and high-molecular weight.
Similar experiments with epithelio-mesen-chymal interactions in the differentiation of kidney, pancreas, salivary gland, thyroid and thymus glands, etc., also suggest this ‘transfilter phenomenon’. The inductive phenomena that occur in all the cases suggest beyond doubt that induction can occur without cell contact and inductive agent is chemical. It involves the transfer of chemicals from the inducing to reacting tissues.
Chemical Nature of Inducing Substances:
The mode of action of inductors and the actual chemical nature of inducing substances are incompletely known. Many chemical substances, like sterols, fatty acids, proteins, glycogen, synthetic carcinogenic agents and even silica particles and dead tissue can induce neural tissue formation.
But these, substances cannot fully imitate the action of normal inductor, because regional organisation of the brain fails to occur by these artificial chemical stimulants. Experiment with the dorsal Up of the blastopore of an amphibian gastrula gives some idea about the chemical nature of organiser.
A portion of the dorsal lip of blastopore from an amphibian gastrula is explanted and cultured in vitro in suitable culture medium. After about 7-10 days, all the cells of the explant are eliminated leaving only the medium in which the explant was cultured.
The medium is filtered by millipore filter paper. The medium is called the “conditioned medium”. A small fragment of embryonic ectoderm, excised from early gastrula, was put in the conditioned medium.
After about one to three weeks, the ectoderm cells formed neural and pigment cells. Thus induction is caused in the absence of inducing cells. This fact indicates that the cells of the dorsal lip of blastopore have certainly liberated the inducing substances directly into the culture medium which have caused neural induction.
Spectrophotometric and electrophoretic analyses indicate that the inducing substance in the medium as the macromolecules—predominantly nucleo- proteins. Now the question arises whether the protein moiety or RNA part of nucleo- proteins is more effective as inducing agent.
Digestion of RNA part by ribonuclease (an enzyme which digests the RNA) does not reduce the inductive capacity to a considerable extent, but similar treatment with proteolytic enzymes (pepsin or trypsin) which digests the protein component reduces the occurrence of neural inductions in about 80% cases.
This experiment suggests to the contention that the protein part of the nucleoprotein is more effective inducing agent.
Biological Organization of Organizer:
Extensive experimentations have been performed to gain an insight into the organization of the organizer, particularly in amphibian development. The dorsal lip of blastopore (chordamesoderm) acts as the primary organizer. Fig. 5.23 indicates that the organizer is not a homogeneous system. The chordamesoderm of a neurula stage is used as the implanted material.
The anterior portion used as graft into the blastocoel induces a head, while the posterior part as a graft produces a secondary trunk and tail. Because of this, the anterior portion is called the head organizer and the posterior part is designated as the trunk organizer.
This differential action of the chordamesoderm indicates that the organizer is heterogeneous, but the origin and nature of differences between the different regions are difficult to interpret. There are diverse opinions regarding this aspect. According to one, there are different inductors present in the chordamesoderm, each one is responsible for the expression of a specific part of-the nervous system.
The other theory advocates that there is a single inducing substance in the chordamesoderm and the differential effect is produced by the different concentration of the same substance. But the existence of both qualitative and quantitative differences in the chordamesoderm is the recent view on neural induction.
The chordamesoderm is regarded as a two-dimensional sheet having a pair of gradients. These two gradients are co-ordinated in such a way in the chordamesoderm that a perfect pattern is established. Neural induction is resulted as the outcome of coordinated interaction of two different inducing agents—one is the forebrain inductor and the other is mesoderm inductor.
The fore-brain inductor can induce forebrain, while the forebrain inductor in conjunction with the mesoderm inductor develops both neural and mesodermal structures. Fig. 5.24 shows the quantitative distribution of the two inducing substances.
The forebrain inductor is present in same concentration along the length of the archenteron roof while the mesoderm inductor is present at its highest concentration at the posterior end and gradually diminishes to zero at the anterior end. Combinations of these two inductors are believed to be responsible for specific development in the induced structures.
Successive inductions occur in the development of embryonic structures. The embryo, as such, represents a dynamic system and the positional relation of cells always remains in flux. Occurrence of successive inductions indicates the presence of diverse quality of embryonic induction.
The theoretical interpretations on embryonic induction discussed herein, are not the final answer to the question of the nature and action of the induction, but it provides valuable information on this line.