Gastrulation: Meaning, Mechanism and Methods

In this article we will discuss about:- 1. Meaning of Gastrulation 2. Basic Mechanism in Gastrulation 3. Methods 4. Morphogenetic Movement 5. Different Chordates 6. Analysis of Mechanism.

Contents:

1. Meaning of Gastrulation
2. Basic Mechanism in Gastrulation
3. Methods used to Study Gastrulation
4. Morphogenetic Movement of Cells in Gastrulation
5. Gastrulation in Different Chordates
6. Analysis of Gastrulation Mechanism

---

1. Meaning of Gastrulation:

The blastula passes into the stage called gastrula by the process—Gastrulation. This process is extremely important in the ontogenetic process of an animal, because the blue-print of the future organisation is laid down during this phase.

During this crucial and dynamic process major presumptive organ-forming areas of the blastula become reorganised in a fashion that allows their ready transfor­mation into the fundamental body plan of a species. Gastrulation is essentially a pro­cess of migration of cells from one place to the other in the embryo. Besides movement of cells, considerable nuclear differentiation also takes place.

In almost all animals it results in:

(i) The establishment and diff­erentiation of three primary germinal layers—ectoderm, mesoderm and endoderm,

(ii) The establishment of nuclear differentiation and

(iii) The beginning of the control of genetic factors over development.

---

2. Basic Mechanism in Gastrulation:

The process of gastrulation involves fol­lowing three cellular activities, cell-move­ment, cell-contact and cell-division. All these mechanisms are carried in a nicely co-ordi­nated and integrated way.

Number of fac­tors are believed to be responsible for this coordination, but it has not been possible to pin point the final answer. It is un­deniable that this process is controlled largely by intrinsic factors which are correlated with the external as well as internal conditions.

---

3. Methods used to Study Gastrulation:

The correct observation of incidences during gastrulation was started from the findings of W. Vogt in 1923. Vogt used vital dyes (Janus green and Neutral red) to mark the cells in an early gastrula and noted that cells during gastrulation ac­tually migrate from one place to the other.

The vital dye technique of Vogt resulted into the application of several other me­thods:

(i) Visible differences in the cyto­plasmic particles were used as natural marker,

(ii) Taging of the cells with carbon particles and

(iii) Taging of the cells with radioactive substances.

---

4. Morphogenetic Movement of Cells in Gastrulation:

During gastrulation, cells from one re­gion of embryo move to another to take up their future fateful position. Two terms, emboly and epiboly which are quite opposite in their meanings, are generally applied to explain the process of movement.

Em­boly means the throwing in or insertion of cells and epiboly signifies the extending upon. The movement of cells establishes a particular form and involved in organ formation in embryo—so this movement is designated as the morphogenetic movement. Fig. 5.15 shows the movement of cells in gastrulation.

Fundamentally, the morphogenetic movement is similar but the details of the process vary greatly.

Following types of cells movement occur:

Epiboly:

It involves the extension along the anteroposterior axis and peripheral divergence.

Emboly:

The inward movement of cells is classified into different types depending on the behaviour of migrating cells.

These are:

(i) Invagination:

It denotes the infold­ing of a layer of cells to form a cavity en­circled by infolded cells. Generally in the gastrulation of Amphioxus and frog, the wall of the blastoderm is pushed inside the blastocoel. This creates a new cavity called the archentecon which communicates with the exterior by a blastopore.

This process of inpushing goes on and the inpushed layer forms the walls of the cavity. The archenteron (or primitive gut) completely obliterates the blastocoel.

(ii) Involution:

It implies the inward lotation of cells as seen in the gastrulation of amphibian and avian eggs. From one end near the edge of the blastoderm, the cells begin to move inwards to form the inner lining of the blastoderm.

(iii) Convergence:

It means the movement of cells to a particular region of the gastrula. In amphibian egg, the migration of cells to the external edge of the blastoporal lip is designated as convergence. The same phenomenon of convergence of cells is seen in the formation of primitive streak in chick embryo.

(iv) Divergence:

This pheno­menon is opposite to convergence, when involuted cells diverge to take up their future positions inside the gastrula.

(v) Infiltration:

During this process, cells of the blastoderm infiltrate near the bottom of the blastocoel to form a second layer as seen in the gastrulation of chick.

(vi) Delamination:

This is a process of separation of a group of cells from others to form discrete cellular masses.

(vii) Extension:

The elonga­tion of presumptive areas after they have moved inside the embryo is called the extension.

(viii) Cell proliferation:

It means the increase in the number of cells during gas­trulation.

(ix) Concrescence:

It is similar to convergence. The cells from two sides mig­rate anteriorly along one axis, but in con­vergence the cells from two sides unite together and then move anteriorly.

The above terms are coined for the con­venience of analysing the events in gas­trulation. Recent observations have estab­lished that it is essentially a phenomenon of integration. It was, therefore, felt necessary to understand the whole process for a mean­ingful comprehension of individual event.

---

5. Gastrulation in Different Chordates:

i. Amphioxus:

The blastula of Amphioxus contains the potential endodermal cells at the vegetal pole, i.e., hypoblast which forms the floor of the blastula. The presumptive organ forming cells (i.e., notochordal, me­sodermal, epidermal, etc.), form the epiblast.

The epiblast constitutes the roof of blastula. The blastocoel is large. The dorsal crescent (presumptive neural and notochordal cells) lies in the future dorsal lip region of the blastopore while the ventral crescent (meso­dermal area) occupies the ventral lip.

With the onset of gastrulation, an in­crease of mitotic activity is observed in the dorsal and ventral crescent regions. With the activity of the different cells, the endo­dermal plate invaginates into the blasto­coel. During this process of invagination, the dorsal portion moves at a faster rate to touch a point which marks the anterior end of the developing embryo.

The noto­chordal cells, occupying the mid-dorsal re­gion of the blastopore, involute and occupy a mid-dorsal position in the developing archenteron. Then the ventral crescents gradually converge on either side of the notochordal cells. Thus the roof of the archenteron is composed of mesodermal and notochordal cells.

This process of eraboly is accompanied by epiboly when the ectodermal and neural cells extend along the antero-posterior direction. The exten­sion of ectodermal cells and the prolifera­tion, involution and infolding of presump­tive endodermal, notochordal and meso­dermal cells result in the formation of a double-layered embryo (Fig. 5.17). The external layer forms the ectoderm.

The internal layer has a dorsomedian area of notochordal cells with two bands of meso­dermal cells. The rest of the inner layer is formed of endodermal cells. Rapid cell proliferation, accompanied by emboly and epiboly causes an anteroposterior elonga­tion of the gastrula.

As the developing gastrula elongates in the anteroposterior direction, the ventral crescent is gradually shifted dorsalward along the inner lateral side of the blastoporal lip. As a result of convergence, the mesoderm comes to lie on the two sides of the notochordal material at the dorsal blastoporal lip.

At the end of gastrulation the blastopore becomes smaller and is closed by ectodermal overgrowth. A neuroenteric canal is formed between the archenteron and developing neural tube.

Mesoderm differentiation:

The transformation of the neural plate to form the neural tube is associated with the formative of a shallow groove on either dorsolateral walls of archenteron. The cells forming these two grooves are smaller than other cells. The grooves become deeper and their edges come together.

Such fusion results in the separation of a solid notochordal rod along the mid-dorsal line. These two lateral grooves become divided by transverse partitions into enterocoelic pouches which grow between the endoderm and ectoderm (Fig. 5.17).

The cavities of these pouches retain their connection with the archenteron at the beginning which become subsequently lost. As a conse­quence paired hollow blocks of mesodmal cells are formed. Formation of hollow mesodermal blocks is observed only in the first two pairs of somites.

The posterior entcrococlic pouches are pinched off as solid blocks of mesodermal cells within which coelomic cavities are formed anew. This process is observed upto fourteenth pairs of somites. In the rest of the posterior segments, the two halves of the original folds meet to form a solid band of cells extending up to the blastopore. The meso­dermal somites differentiate from the late­ral bands.

On the basis of origion the mesoderm is divided into:

(a) Gstral meso­derm and

(b) Pristomial mesoderm.

The gastral mesoderm develops from the enterocoelic pouches, while the peristomial mesoderm differentiates from the lateral bands.

The somites or segmental mesoderms gradually grow ventrally on either side until they meet in the midventral line below the alimentary canal. The meso­dermal sheet becomes double-walled en­closing coelome within themselves. The lateral plate mesoderm becomes thus splitted into (i) somatic mesoderm in associa­tion with ectoderm and (ii) splanchnic mesoderm in association with the endoderm.

ii. Frog:

In late amphibian blastula, the presumptive organ forming areas are oriented around the blastocoelic cavity.

The hypoblast is situated at the vegetal pole, while the epiblast is located at the animal pole. In the epiblast the noto­chordal cells, neural plate and epidermal areas are situated along the anteroposterior axis of the blastula with the notochordal cells located at the most posterior position.

At the end of the cleavage all the blasto­meres remain stationary and none of them have shiftecf from its original position. But at the onset of gastrulation a great mass migration started to occupy their definite position in the developing embryo. Gastru­lation begins with the appearance of a small cleft-like invagination at one side and just above the grey crescent (Fig. 5.18).

This cleft-like invagination is crescent- shaped and represents the dorsal lip of the blastopore. As gastrulation progresses the crescent-shaped cleft continues to expand to assume a semicircular appearance, then becomes horse-shoe-shaped and finally forms a ring. This ring represents the blastopore. The blastopore becomes the focal point for gastrulation activities.

Migration of cells inside the gastrula starts along the newly-formed dorsal lip of blastopore and this inward pushing is caused by the endodermal cells which are folded inward (Fig. 5.19) and forward towards the future anterior end of the em­bryo. The upper margin of the blastopore is called the dorsal lip of the blastopore and the lower edge is designated as the ventral lip of the blastopore.

As invagina­tion expands within the blastocoel, the prechordal plate cells from the upper part of the dorsal side move inward. The new cavity thus produced is called the archen­teron which communicates to the exterior by the blastopore. With the further ad­vancement of invagination, the archen­teron continues to expand by obliterating the blastocoel.

The inward moving cells form a new border beneath the outer cells. The roof of the archenteron consists of the involuted layer which includes the endo­derm and mesoderm. Beyond this layer lies the ectodermal layer. The floor of the archenteron is made up of a layer of endo­dermal cells, the derivatives of the large yolk cells which were located in vegetal hemisphere of blastula.

When the inward movement of the cells is in progress through the dorsal lip, an­other type of movement occurs on the outer side. The pigmented cells of the ani­mal hemisphere started to enclose the macromeres of vegetal hemisphere. After completing the enclosure, the outer cells reach up to the ventral lip of it.

A small mass of macromeres remains uncovered for a while and acts as a plug of the blastopore. It is called yolk plug. At this stage, embryo is made up of two distinct strata, each of which is composed of many layers of cells.

Differentiation of three primary germ-layers:

The blastula of frog is mono-layered which in course of gastrulation becomes converted into a triploblastic stage, i.e., three cell-layered. These three layers are designated as the primary germ-layers (embryonic ectoderm, embryonic meso­derm and embryonic endoderm). All the organs of the developing embryo develop from these three primary germ-layers.

(a) Ectoderm:

The pigmented cells of the animal pole, which spread to enclose the macromeres of the vegetal hemisphere be­come differentiated into ectoderm.

(b) Endoderm:

The dorsal and lateral sheets of cells which form the roof of the archenteron represent the endoderm as well as mesodermal material. Upon com­pletion of gastrulation, the roof and sides of the archenteron become lined by a single layer of endodermal cells which have differentiated from the involuted several celled thick archenteron roof.

(c) Mesoderm:

As soon as the endodermal sheet becomes separated dorsally and late­rally from the involuted cells, mesodermal sheet is being formed between the endo­derm and ectoderm. The mesodermal sheet starts its differentiation anteriorly and then proceeds gradually backwards.

The mesodermal sheet is divided into two halves by a narrow band of median cells which develop into notochord. Laterally the mesodermal sheets grow downward and finally the right and left mesodermal sheets unite in the mid-ventral line to become a continuous mesodermal sheet.

The three layers thus formed are ectoderm, meso­derm and endoderm. It is the special fea­ture in amphibian development that gas­trulation results into the formation of meso­derm first and then the endoderm.

iii. Chick:

The blastoderm has a central area free from yolk which is called the area pellucida, while the germ-wall with the adhering yolk constitutes the area opaca. In course of development, the blastoderm becomes converted into a double-layered structure—the upper one is the epiblast and the lower layer is called the hypoblast.

The space between these two layers is called the blastocoel while the space below the hypoblast is the primordial archenteron.

The epiblast contains presumptive ecto­dermal and neural areas at the anterior portion while the posterior half comprises of presumptive notochordal and mesoder­mal cells. The hypoblast transforms into the endoderm and the epiblast is converted into ectoderm and mesoderm (Fig. 5.20).

At the initiation of gastrulation, the hypoblast cells from the posterior end start migrating towards the anterior end of the embryo along the median line. Immedia­tely after the inauguration of the movement in the hypoblast, the cells of the epiblast overlying the migrating hypoblast move downward towards the hypoblast.

These involuted cells occupy a position between the epiblast and hypoblast and migrate to the lateral and anterior ends bet­ween the epiblast and hypoblast. Movement of cells in the blastoderm of chick during gastrulation has been studied by Spratt (1946) by carbon particle tech­nique.

With the activities of the epiblast and hypoblast, the presumptive mesoderm cells from the posterior half of the epiblast move posteriorly and converge from the lateral sides towards the median line. These converging cells begin to accumu­late at the posteromedian border of the area pellucida as a raphe-like thickened structure. This marks the appearance of the primitive streak (Fig. 5.21).

The migratory cells after coming to this region move inwards and migrate anteriorly and laterally. The migratory cells of the epiblast move downward and occupy the position between the epiblast and hypo­blast. These cells then diverge anteriorly and laterally as a broad middle layer of mesodermal cells along the primitive streak.

Daring its forward movement, it appro­aches the presumptive notochordal areas. With the anterior movement the streak also starts to move backward. The primi­tive streak is fully formed at about 18-19 hours after incubation. Gradually the area pellucida changes from a round to pear-shaped appearance.

The primitive streak represents the posterior region of the deve­loping embryo and the embryo proper develops anterior to it. It is also an area of cell proliferation arid rapid growth. The primitive streak becomes very con­spicuous in early embryonic life. It consists of a groove (Primitive groove) which is flanked on both the sides by two ridges (Primitive ridges).

It terminates anteriorly in a primitive pit, and posteriorly in a primitive plate. Immediately anterior to the primi­tive pit (which represents the defunct neuroenteric canal) lies an elevation, Hensen’s node or Head process.

In this area the mesoderm becomes thickened and pro­jects from the primitive streak. With the formation of the Hensen’s node, the primi­tive streak regresses posteriorly arid the major organ forming areas become well established! A groove appears on the outer surface of the head process and the two folds unite to form a tube.

The forma­tion and closure of the groove continue posteriorly. The entire process may be compared with the action of a zipper. As die closure of the groove comes to the posterior end, the backward move­ment ceases leaving an opening at the posterior end.

In the gastrulation of chick, the meso­derm differentiates lastly from the epiblast by the process of involution, elongation, expansion and extension. The hypoblast gives rise to endoderm and the epiblast differentiates into ectoderm and meso­derm.

iv. Mammal:

In the mammalian blastula (Blastocyst) the formative area (germ disc) is restricted at one end. The germ disc is composed of epiblast and hypoblast. In the embryo of pig, the gastrulation activities are observed at two centres—the posterior end forms the primitive streak while the anterior end forms the Hensen’s node.

The behaviour of these portions is almost similar to that observed in the gastrulation of chick. The mesodermal cells from the primitive streak move between the epi­blast and hypoblast and form two wing-­like areas.

The mesodermal cells are divid­ed into:

(a) Embryonic mesoderm confined to the germ disc and

(b) Extra embryonic mesoderm.

---

6. Analysis of Gastrulation Mechanism:

Lillie (1913) established that the surface layer of the egg at first remains plastic but in course of development it loses its plasti­city and becomes rigid. Spemann (1918) found that up to gastrulation, when the eggs are cut into two halves, each will form a complete embryo.

But after gastrulation, each half gives rise to half embryo. He also noticed that the half containing blastopole forms a complete embryo. He came to the conclusion that blastopore plays an important role in gastrulation.

Later he and Mangold (1924) grafted blastopore of one to the gastrula of another and demons­trated that the grafted blastopore influ­ences the host tissue to form embryonic axis. They termed the blastopore as “organiser” and the influence of organiser as “induction”. Spemann’s lead was soon followed by different workers and consi­derable information became available re­garding the nature of organiser.

It may be summarised that induction involves three distinct events:

(i) Evocation,

(ii) Individua­tion and

(iii) Competence.

The first two, evocation and individuation are the pro­perties of organiser and the competence is the feature of the tissues on which the orga­niser acts.

In 1943, Holtfreter, working on the gas­trulation mechanism of amphibian eggs, demonstrated that superficial cells are united by an extracellular surface coat and the beginning of invagination is due to the expansion of certain cells. This expansion according to him is caused by the change of surface tension due to the high pH of blastocoelic fluid.

Though many workers have questioned the findings of Holtfreter, it remained true that initiation of invagi­nation is the property of localized cells. It may be due to local difference of pH or due to differential adhesiveness of the cells.

The work done to explore the nature of involution and epiboly also explained that the entire process is due to the nature of participating cells. It was demonstrated that the cells which are more adhesive are less mobile and on the contrary more mo­bile cells are less adhesive. Once this was understood attempts were made to explain the mechanism of gastrulation in terms of cellular adhesibility and cellular mobility.

In 1955, Townes and Holtfreter exa­mined the interaction of different cell layers in amphibian gastrulae and demons­trated:

(a) That endoderm cells are less adhesive than mesoderm,

(b) That outer ectoderm is less adhesive than inner ecto­derm and

(c) Mesoderm is less adhesive than inner ectoderm but more adhesive than endoderm.

Basing on this contention Stainberg (1964) proposed that disposition of different layers in a gastrula depends upon the adhesive nature of the cells. Outer ecto­derm being less adhesive stays outermost. Mesoderm being less adhesive than inner ectoderm but more adhesive than endo­derm remains in between the two.