The early development of Xenopus: Cleavage and Gastrulation

Zoology Lesson : The early development of Xenopus - Cleavage and Gastrulation Lesson Developer: Dr Meena Yadav College/Department: Maitreyi College, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation

TABLE OF CONTENTS  INTRODUCTION  CLEAVAGE  Experiment by Nieuwkoop  GASTRULATION  Fate maps in Xenopus  Cell movements during gastrulation  Next phase of gastrulation  Midblastula transition  Change in position of the blastopore  Invagination and involution:  Experiment by Johannes Holtfreter  Dorsal mesoderm and its positioning  Involuting mesoderm  Epiboly of Ectoderm  PATHWAYS OF MESODERMAL MIGRATION  SUMMARY  EXERCISE/PRACTICE  GLOSSARY  REFERENCES  SUGGESTED READINGS  USEFUL WEB LINKS

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The early development of Xenopus: Cleavage and Gastrulation

INTRODUCTION Before World War II, embryological research came to an almost halt as eggs were not available readily and they had to be collected from wild. In 1930‟s it was discovered that female Xenopus will ovulate, if injected with urine from a pregnant human female. In 1940‟s and 1950‟s, it was the only available pregnancy test and many hospitals kept Xenopus. But these frogs are very fast in escaping. They, thus, escaped and their population increased in the nearby areas like California, South America and also in colder areas like South Wales. From 1950‟s onwards, Xenopus became the model organism of choice for studying developmental biology as its eggs were readily available. Thus, Xenopus, the African clawed frog, became an ideal model organism for embryological studies, even for the molecular level studies. The size of the eggs and embryo of Xenopus is quite large which makes it suitable for microsurgical studies. Xenopus cannot be used for studies in genetics as females take approximately a year to become sexually mature and thus breeding experiments cannot be carried out. Moreover, Xenopus is allotetraploid i.e. it has four copies of each gene. The female Xenopus lays many transparent eggs, making it easier to observe developmental stages. These frogs are very easy to breed, rear and can be kept in captivity for as long as 20 years. Two species of Xenopus are readily used in the biomedical research and developmental studies i.e. Xenopus laevis and Xenopus tropicalis. Following features make Xenopus eggs and embryos suitable models in biomedical research: i. Due to large size of the eggs and embryo it is easy to inject a variety of materials into them ii. Their embryos can tolerate extensive manipulations like dissections, transplantations etc iii. Since the fate of the cells in known during early embryonic development, so it is easy to carry out targeted gene knockout, knock down and over expression studies. iv. They are ideal for biochemical studies. v. They are also used for studies of ion transport and channel physiology vi. Their oocytes are used to assay environmental toxicology vii. The cell free extracts can be used to study fundamental aspects of cell and molecular biology

Value Addition: Do you Know? Heading Text: Comparison of two common species of Xenopus Body Text: Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation

Ploidy Haploid Genome size Optimal temperature Adult size Egg size Brood size Generation time

Xenopus laevis Allotetraploid 18 chromosomes 3.1 x 109 bp 16-22º C 10 cm 1-1.3 mm 300-1000 1-2 years

Xenopus tropicalis Diploid 10 chromosomes 1.7 x 109 bp 25-30º C 4-5 cm 0.7-0.8 mm 1000-3000 4 months

Source: http://www.xenbase.org/anatomy/intro.do

Value Addition: Do you Know? Heading Text: About Xenopus laevis Body Text: The skin of Xenopus laevis produces maganin, a substance with antibiotic, antifungal, antiviral and antiparasitic properties, which is useful for the survival of frog in its native habitat, stagnant ponds. It was also the first vertebrate to be cloned.

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The early development of Xenopus: Cleavage and Gastrulation

Source: http://guides.library.harvard.edu/content.php?pid=237931&sid=2016930

CLEAVAGE The event i) ii) iii) iv) v)

of fertilization in frogs leads to several changes in the egg: The second meiotic division is completed Cytoplasm of the egg rotates to 30º with respect to both the poles. In some amphibians like Xenopus, the rotation of the cytoplasm establishes a gray colored area around the equator of the egg called gray crescent. Gray crescent appears opposite to the point of sperm entry The haploid nuclei fuse to form diploid nucleus

The Xenopus egg is light colored at the lower end which is called as vegetal pole, while its upper end is dark colored and is called animal pole. Both the poles contain different set of mRNA and other cell components, which are distributed accordingly during the subsequent cleavage divisions. The vegetal pole contains mRNAs for regulatory molecules like VegT, signal proteins of TGFβ superfamily and proteins of Wnt signaling pathway. The first cleavage division in frogs occurs about 75-100 minutes after fertilization, depending on the temperature. In most of frogs and salamander embryos, cleavage is holoblastic and radially symmetrical. In amphibians, the eggs contain more of yolk which is concentrated in the vegetal hemisphere and thus interferes with the cleavage. The first cleavage division, thus, begins at animal pole and slowly extends down towards the vegetal pole (Figure 1). It has been observed in axolotl, that the cleavage furrow extends at a rate of approximately 1 mm per minute. However, once the furrow crosses the grey crescent area, the rate drops to 0.02-0.03 mm per minute.

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The early development of Xenopus: Cleavage and Gastrulation

Figure 1: The first two cleavages in Xenopus egg Source: Author Even when the first cleavage is under process towards the vegetal pole, the second cleavage starts at the animal pole. The delay in the completion of the first cleavage is due to the yolky cytoplasm. The second cleavage is at right angles to the first one and is also meridional. The third cleavage is, however, equatorial. But due to large yolk in the vegetal hemisphere, the cleavage is not actually at the equator but is displaced towards the animal pole. This cleavage produces four smaller cells at the upper side (i.e. animal hemisphere), called micromeres and four larger cells towards the lower side (i.e. vegetal hemisphere), called macromeres (Figure 2). This establishes two regions in the embryo – one region in the animal hemisphere consisting of rapidly dividing micromeres and the other region in the vegetal hemisphere consisting of slowly dividing macromeres. As the cleavage divisions continue, the animal hemisphere becomes packed with cells while the vegetal hemisphere contains comparatively lesser number of cells, which are laden with yolk.

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The early development of Xenopus: Cleavage and Gastrulation

Micromeres

Macromeres

Figure 2: Morula of Xenopus showing micromeres and macromeres Source: Author In amphibians, the ball of cells (16-64 cells) accumulated after initial cleavage divisions is known as morula. The blastocoel appears at the 128 cell stage and then the embryo is called as blastula. However, the appearance of the blastocoel can be traced back to the first cleavage, as demonstrated by Kalt (1971). He said that the first cleavage furrow widens to create a small intercellular cavity in the animal hemisphere which is not visible from outside as it is closed by tight intercellular junctions. This cavity widens during subsequent cleavage divisions and is later known as blastocoel (Figure 3).

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The early development of Xenopus: Cleavage and Gastrulation

Ist Cleavage furrow

II

Animal hemisphere

III

I

Gray crescent Vegetal hemisphere (i)

(ii)

(iii)

Blastocoel V

III IV

IV

V IV

Blastula (vii)

Morula (vi)

(v)

(iv)

Figure 3: The stages of cleavage divisions till the formation of blastula Source: Author The two major functions of blastocoel are: i. migration of cells during gastrulation, ii. prevents premature interaction of cells above and below it

Experiment by Nieuwkoop He took embryonic newt cells from roof of blastocoel in animal hemisphere and put it adjacent to yolky cells from vegetal hemisphere, taken from base of the blastocoel. He found that cells taken from the roof differentiated into mesodermal tissue instead of ectoderm. This shows that the vegetal cells, precursors of mesodermal tissue, influence the differentiation of the adjacent cells from the animal hemisphere to differentiate into mesodermal tissue. Thus, blastocoel prevents the contact of the cells in two hemispheres, which are fated to give rise to different organs of the body. The micromeres in the animals hemisphere and macromeres in the vegetal hemisphere remain in contact with each other respectively by cell adhesion molecules (CAMs). One of the most important CAMs is EP-cadherin, whose mRNA exists in the oocyte cytoplasm. If this mRNA is destroyed, then the EP-cadherin is not synthesized and the blastocoel is obliterated due to non-adhesin of the blastomeres.

INSERT THE PPT Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation

Nieuwkoop's Center Source : faculty.mwsu.edu/biology/jon.scales/courses/DevBiol/.../vert_dev_2.ppt

GASTRULATION The study of amphibian gastrulation is one of the oldest areas of embryology. But, the knowledge about the mechanisms keeps on upgrading from time to time due to newer research methodologies. The process of gastrulation is complex in amphibians because each species employs a slightly different mechanism. Gastrulation initiates approximately 10 hours after fertilization. Most of the study has been performed on Xenopus laevis in this regard and has been discussed in the following sections

Fate maps in Xenopus The amphibian blastula must engage itself in the movement of the cells for gastrulation such that the cells destined to form endodermal organs are brought inside, the cells that will form ectoderm surround the embryo and the mesodermal cells are placed in between the ectoderm and endoderm. Løvtrup (1975), Landstrom and Løvtrup (1979) and Keller (1975,1976) have shown, by using vital dye staining, that the location of the cells in the embryo determine their fate (Figure 4). It has been seen that in Xenopus, the mesodermal cells lie deep in the blastula while the ectodermal and endodermal cells lie superficially, on the surface of the embryo. Most of the precursors for notochord and other mesodermal tissues are located underneath in the equatorial region of the embryo. In urodeles and in some frogs, other than Xenopus, more precursors of notochord and mesoderm lie on the surface of the embryo. In amphibians, the unfertilized egg has polarity i.e. it has distinct animal and vegetal poles. Thus, the germ layers can be mapped even before fertilization occurs. The area around the animal hemisphere will give rise to ectoderm, the area of vegetal hemisphere will form endoderm (gut and associated organs) and the area around the internal cytoplasm near the equator will form the mesodermal cells. This general pattern of fate of the areas in the oocyte is result of a transcription factor VegT, and the paracrine factor Vg1. In Xenopus, the mRNAs for these factors are located in the cortex of the vegetal hemisphere and are allocated proportionally to the macromeres during cleavage. Zhang et al (1998) depleted maternal VegT protein in early embryos by using antisense oligonucleotides. As a result, the embryos lacked the footprint of a

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The early development of Xenopus: Cleavage and Gastrulation

Figure 4: Fate map of Xenopus blastula Source: http://www.devbio.biology.gatech.edu/?page_id=345 CC

Click on the link http://www.xenbase.org/anatomy/static/xenbasefate.jsp and learn about the fatemap of Xenopus in an interactive manner where you Move cursor over early blastomeres to see their future positions.

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The early development of Xenopus: Cleavage and Gastrulation

Value Addition: Do you Know? Heading Text: Life Cycle of Xenopus laevis Body Text:

Source: http://www.mun.ca/biology/desmid/brian/BIOL3530/DEVO_03/ch03f03.jpg

normal fate map. The animal third (future mesoderm) produced only ventral epidermis, while marginal cells (future mesoderm) produced epidermal and neural tissues (Figure 5). The vegetal third (future endoderm) produced both ectoderm and mesoderm. The embryos lacking functional Vg1, lacked endoderm and dorsal mesoderm as demonstrated by Joseph and Melton (1998).

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The early development of Xenopus: Cleavage and Gastrulation

Surface of animal hemisphere: Cells of ectoderm (skin & nerves)

Ventral epidermis

Animal-vegetal axis

Mesoderm Epidermis & Neural tissue

Vg1 Surface of vegetal hemisphere: Cells of endoderm (Gut & associated organs)

Ectoderm & Mesoderm VegT

Fate map of a treated embryo (depletion of VegT)

Fate map of a normal embryo

Figure 5: Lack of normal fate map in the embryos lacking VegT Source: Author

Cell movements during gastrulation Gastrulation in frog embryos is initiated at the future dorsal side of the embryo, just below the equator in the region of gray crescent. In this region, the cells invaginate to form a slit like aperture called blastopore. The shape of these cells changes dramatically such that the main body of each cell is displaced towards the inside of the embryo but maintains its contact with outside surface by a slender neck. These bottle cells line the archenteron. Unlike in sea urchin, where gastrulation begins at most vegetal region, in frogs, the gastrulation begins at the marginal zone i.e. the zone surrounding the equator in the balstula. At the equator, the endodermal cells are not as large or yolky as the most vegetal blastomeres.

Next phase of gastrulation: In this phase, there is involution of the marginal zone cells while the cells at the animal hemisphere undergo epiboly and converge at the blastopore. When the migrating marginal cells reach the dorsal lip of the blastopore, they turn inward and travel along the inner surface of the outer animal hemisphere cells. Thus, the cells of the lip of blastopore keep constantly changing. The first cells of the dorsal lip of the blastopore are the bottle cells, which later become the pharyngeal cells of the foregut. These bottle cells then pass into the interior of the embryo. The next cells to form the lip of the blastopore are the cells that will involute into the embryo to become the prechordal plate (they are precursor of the head mesoderm). The next cells to line the blastopore Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation lip are the chordamesoderm cells which also involute into the interior of the embryo. These cells form the notochord , that acts as a transient mesodermal “backbone” and plays an important role in patterning of the nervous system. As the new cells keep on entering the interior of the embryo, the blastocoel is pushed to the opposite side of the dorsal lip of the blastopore. Further, the blastopore lip expands laterally and ventrally. The widening of the blastopore lip leads to the formation of lateral lips and a ventral lip, over which additional mesodermal and endodermal precursor cells pass. The blastopore forms a ring around the large endodermal cells after the formation of the ventral lip. The endodermal cells remain exposed on the vegetal surface. This patch of the endoderm is called yolk plug and is later internalized. At this point of the gastrulation, all the endoderm cells have been internalized, ectoderm has encircled the embryo and the mesoderm lies in between the two layers.

Midblastula transition The events of the gastrulation are guided by the inputs from the genome. So, the genes have to be transcribed. In Xenopus, the genes ar not transcribed until the cells are in the late twelfth cell cycle. At that point, different genes are transcribed in different cells. The blastomeres also become capable of movement. This change is known as midblastula transition. At this time, different transcription factors become active in different cells leading to differential response of the cells. For example, the vegetal cells become endoderm and these cells in turn secrete certain factors that influence the cells above them to become mesoderm.



Change in position of the blastopore The point of the sperm entry as well as the vegetal cells play a role in determining the position of the blastopore. The cytoplasmic movements in egg, immediately after the sperm entry, are controlled by the sperm microtubules, which empower the vegetal cells to induce the blastopore in the mesoderm above them. The protein Dishevelled is transported by the microtubules to the future dorsal side. This protein is a downstream component in the Wnt signaling pathway. The region in which the protein Dishevelled is present, start expressing dorsal-specific genes. The region of cells opposite the point of sperm entry forms the blastopore and becomes the dorsal portion of the body. The unfertilized frog egg is radially symmetrical about the animal-vegetal axis, but the fertilized egg has a dorsal-ventral axis. The internal cytoplasm of the fertilized egg remains oriented due to gravity and the dense yolk while the cortical cytoplasm rotates 30º towards the animal hemisphere i.e. towards the point of sperm entry. The fertilized egg, thus, becomes bilaterally symmetrical. Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation Fluorescence microscopy has shown that in early embryos, the cytoplasm of presumptive dorsal cells differs from that of the presumptive ventral cells. These cytoplasmic movements activate the cytoplasm opposite to the point of sperm entry and thus, initiates the gastrulation in the vegetal hemisphere. The side of the sperm entry becomes the future ventral surface of the embryo while the opposite side, where the process of gastrulation starts becomes the future dorsal surface (Figure 6). If the cortical cytoplasmic movements are blocked, there is no development of the dorsal side and the embryo dies.

Animal pole

Sperm Future ventral surface

Gastrulation begins Future dorsal surface Vegetal pole Figure 6: Fate of dorsal-ventral axis in frog embryo Source: Author

The movements of the egg cytoplasm are not totally dependent on the sperm entry, but the sperm determines the direction of the rotation. If the egg is artificially activated, the cortical rotation occurs at the correct time but the direction of the movement is not defined. This problem can be over come by mechanically redirecting the spatial relationship between the cortical and internal cytoplasm. For example, if the Xenopus egg is turned 90º, so that the point of sperm entry faces upward, the cytoplasmic movements become such that the gastrulation starts on the same side as the sperm entry. Black and Gerhart (1985, 1986) demonstrated that it is possible to initiate two gastrulations by giving two stimuli i.e. sperm entry and mechanical rotation of the egg (Figure 7). They allowed the sperm-directed rotation to occur but them immobilized the eggs in gelatin and centrifuged them gently so that the cytoplasm moves towards the point of sperm entry. When these eggs were later allowed to develop in normal water, a second site of gastrulation also developed, thus producing conjoined twin larvae. They hypothesized that this was the result of two areas of interaction: one due Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation to normal cortical rotation caused by cytoplasmic interactions in the vegetal region of the cell, and the other where the centrifugation-driven cytoplasmic interactions in the vegetal area.

Animal pole

Sperm Future ventral surface

Second gastrulation begins

Immobilize the eggs on gelatin Centrifuge to redistribute cytoplasm

Gastrulation begins Future dorsal surface Vegetal pole

First gastrulation continues

Figure 7: Experiment of Black and Gerhart Source: Author It is the cortical rotation that causes the vegetal cells to induce the cells above them to start gastrulation.

Experiment of Gimlich and Gerhart (1984): They irradiated the 64-cell Xenopus embryo by UV rays, such that it fails to properly initiate gastrulation. After this, they transplanted three vegetal blastomeres, from the normal embryo to the UV-irradiated embryo, on the opposite side of the point of sperm entry. The transplanted blastomeres were seen to induce the formation of dorsal lip of blastopore and a complete dorsal axis. (Figure 8A). Interestingly, when these three blastomeres (precursors of prospective dorsal lip region) were transplanted in an unirradiated embryo, they can induce a secondary blastopore on the ventral side of the normal embryo (Figure 8B).



Invagination and involution:

The first sign of the amphibian gastrulation is when the marginal endodermal cells on the dorsal surface of the blastula sink into the embryo. The outer or apical surfaces of these endodermal cells contract while their inner or basal ends expand. These cells elongate towards the inner side of the embryo to gain the characteristic „bottle‟ shape and are called ‘bottle cells’. These cells have an active role during early cytoplasmic movements in Salamander gastrulation.



Experiment by Johannes Holtfreter (1943, 1944): Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation

He isolated bottle cells from early salamander gastrulae and found that these cells could attach to the glass coverslips and could motivate other movement of neighboring cells. In another experiment, when dorsal marginal zone cells (precursors of dorsal lip of blastopore) were isolated and placed on inner prospective endoderm tissue, they formed the bottle cells and sank beneath the surface of inner endoderm. As they sank further, they laid the foundation of the early blastopore. These experiments proved that the dorsal marginal zone cells have the innate ability to invaginate deep into the endoderm.

Normal embryo

UV-irradiated embryo

Transplant three vegetal blastomeres Normal tadpole

Normal tadpole Embryonic piece lacking body axis

Figure 8 (A): Transplantation experiment by Gimlich and Gerhart Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation Source: Author

R.E. Keller (1981) and Keller et al (1988) showed that in Xenopus, the bottle cells may play an important role in initiation of the sinking of the marginal cells as they become bottle shaped, but they are not required for the later events of gastrulation. The dramatic change in shape of marginal cells is needed as it produces the bottle shaped cells and produces a slit-like blastopore. However, after the start of these movements, bottle cells are not required for gastrulation. Even if we remove the bottle cells, further events of gastrulation continue unperturbed.

Normal embryo

Normal unirradiated embryo

Transplant three vegetal blastomeres

Normal tadpole

Embryo with two gastrulation sites and body axes

Figure 8 (B): The transplantation experiment by Gimlich and Gerhart Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation Source: Author

Further movement of cells in the embryo is as a result of involution of subsurface marginal cells. The inward movements of the endodermal vegetal cells reposition the prospective pharyngeal endoderm adjacent to the roof of the blastocoel. So, the prospective pharyngeal endoderm comes to lie ahead of the migrating mesoderm. After this, the cells migrate along the basal surface of the blastocoel roof. The superficial layer of the marginal cells is pulled inward and forms the endodermal lining of the archenteron because it is attached to the actively migrating deep cells. At this stage, if the deep involuting marginal zone (IMZ) is removed and replaced with the animal region cells that do not undergo involution, the formation of the archenteron is stopped (Figure 9).

IMZ

Involuting subsurface marginal cells Bottle cells

Blastopore

Archenteron

Figure 9: Formation of archenteron by involution of marginal zone cells Source: Author The dorsal lip of blastopore is an identity in itself. If the cells of dorsal lip of blastopore are excised from the early frog embryo and transplanted into another embryo at any other position, the recipient embryo starts gastrulation at two places: one at its own point and the other at the point where the transplant is present. Thus, it results in formation of two embryos i.e. twins. Thus, dorsal lip of blastopore is a Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation centre of signaling which coordinates gastrulation as well as patterning in its surrounding cells. Thus, it is also known as Organizer or Spemann’s organizer.

Value addition: Heading text: Hans Spemann Body text: Hans Spemann (1869 1941) A German embryologist who worked extensively on amphibian development and was the discoverer of the organizer region (or primitive node) the controller of gastrulation. He received the 1935 Nobel Prize in Physiology or Medicine for his discovery of the effect now known as embryonic induction, an influence, exercised by various parts of the embryo, that directs the development of groups of cells into particular tissues and organs. Source: https://upload.wikimedia.org/wikipedia/commons/thumb/8/82/Hans_Spemann _nobel.jpg/180px-Hans_Spemann_nobel.jpg

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Dorsal mesoderm and its positioning

On the dorsal side, the process of involution is led by pharyngeal endomesoderm and the prechordal plate. The migration of these tissues occurs to the anterior side beneath the superficial ectoderm. After this, the next tissues to migrate, to enter the dorsal blastopore lip, are precursors for notochord and somites. The lip of the blastopore expands to develop dorsolateral, lateral and ventral sides. At the same time, prospective heart mesoderm, kidney mesoderm, and ventral mesoderm enter into the embryo. The IMZ is originally several layers thick. Before the involution of the IMZ cells through the blastopore lip, several layers of IMZ cells intercalate radially to form one thin but broad layer. The intercalation extends the IMZ towards the vegetal end. Simultaneously, the superficial cells divide, flatten and spread. When the deep cells reach the blastopore lip, they involute into the embryo and start another set of intercalation. This intercalation causes a convergent extension along the mediolateral axis. This extension integrates several mesodermal streams to form a long, narrow band. The anterior part of this band starts migrating to the animal cap. Thus, as the mesodermal stream continues to move towards the animal pole, the overlying layer of superficial cells (including bottle cells) is also pulled passively towards the animal pole, thus forming the roof of the archenteron. The radial and mediolateral intercalations of the deep layer of cells may be responsible for continued Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation movement of the mesoderm into the embryo. The convergent extension seems to be driven by two cell adhesion molecules – paraxial protocadherin and axial protocadherin. The paraxial protocadherin is initially found throughout the dorsal mesoderm, but later its expression is turned off in the precursors of notochord. At this time, the axial protocadherin is expressed in the notochordal tissue.

 Involuting mesoderm As the mesodermal migration progresses, the involuting marginal zone continues to narrow and lengthen due to convergent extension. IMZ has two layers as it moves: i) Superficial layer (IMZs): prospective endodermal roof of the archenteron, ii) deep layer (IMZD): prospective mesodermal cells, including those of the notochord. During the middle third of the gastrulation, the expanding mesoderm converges towards the midline of the embryo, which is driven by the continued mediolateral intercalation of cells along the anterior-posterior axis. This further narrows the band. As the gastrulation is coming to an end, the notochord, which was centrally located, now begins to separate itself from the somatic mesoderm on either side of it and it then elongates separately. One reason for this change in position of notochord cells may be due to the different protocadherins in the axial and paraxial mesodermis. This phenomenon seems to be autonomous, since it still occurs if these cells are isolated from the rest of the embryo. During gastrulation, epiboly occurs that causes the animal cap and noninvoluting marginal zone (NIMZ) cells to expand and cover the entire embryo. The dorsal portion of the NIMZ expands more rapidly towards the blastopore and hence, causes the blastopore lips to move towards the ventral side. The mesodermal cells that enter through the dorsal lip of the blastopore, form the dorsal axial mesoderm (i.e. notochord and somites) while rest of the mesoderm enters through the lateral and ventral blastopore lips to create the mesodermal mantle (i.e. blood, kidneys, heart, bones and several other organs). IMZS cells, that line the archenteron roof and subblastoporal vegetal cells, that form the archenteron floor, together form the endoderm.

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The early development of Xenopus: Cleavage and Gastrulation

(A)

(B)

(C)

(D)

Figure 10: Gastrulation in frog embryo (A) (B) (C) (D)

The involution starts at the dorsal lip of the blastopore, and is lead by bottle cells As the involution continues, a new cavity called archenteron starts to be formed As the involution continues, the size of the archenteron keeps increasing and the size of blastocoel keeps decreasing The archenteron takes over as the only cavity in the embryo and is lined by the endodermal layer, the embryo is covered by ectoderm and in between lies the mesoderm. The cavity is not visible from outside and has an opening that is closed by a yolk plug.

Source: https://classconnection.s3.amazonaws.com/975/flashcards/1168975/png/screen_sh ot_2012-03-29_at_15620_am1333000633968.png

Click the link and watch how gastrulation takes place in frog :https://www.youtube.com/watch?feature=player_embedded&v=IjyemX7C_8U

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The early development of Xenopus: Cleavage and Gastrulation

 Epiboly of Ectoderm The ectodermal precursors expand over the entire embryo. Keller (1980) and Keller & Schoenwolf (1977) have shown the changes in the superficial cells and deep cells of the animal and marginal regions by using scanning electron microscopy. The major event that occurs during epiboly in Xenopus is the increase in the number of cells (through cell division) and integration of several layers into one layer. During the early gastrulation events, the number of deep cell layers in animal hemisphere increases after three rounds of cell division. Several cell layers integrate to form one thick layer. The superficial layer expands by cell division and flattening of cells. The changes in the shape of the cells plays important role in the vegetal region than in the animal region. These expansions result in the epiboly of the superficial and deep cells of the animal cap and NIMZ over the surface of the embryo. Majority of the marginal zone cells involute and join the mesodermal cell stream in the embryo. As the ectoderm spreads all over the embryo, it internalizes all the endoderm inside it. This places all the layers in their respective positions i.e. endoderm at the interior, ectoderm at the exterior surface and mesoderm, in between these two layers.

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The early development of Xenopus: Cleavage and Gastrulation

Value Addition: Do you Know? Heading Text: Epiboly of the ectoderm in frogs Body Text:

Fig. Epiboly of the ectoderm A. Morphogenetic movement of the cell migrating into the blastopore and then under the surface B. Changes in the region around blastopore as the dorsal, ventral and lateral lips are formed in succession when the ventral lip completes the circle the endoderm becomes progressively internalized. Source: ILLL in house

PATHWAYS OF MESODERMAL MIGRATION All the movements, which occur during the gastrulation, are precisely controlled. There must be a strong communication cascade for the cells which directs them to move. It has been found that in salamanders, there is a fibronectin lattice, secreted by cells of the blastocoel roof, on which the involuting mesodermal precursor cells migrate towards the animal pole. Just before the gastrulation, the presumptive ectoderm of the blastocoel roof secrets an extracellular matrix, containing fibrils of fibronectin. The mesoderm travels along these fibrils, as if they are the „pathways‟ to be followed. This hypothesis was tested by chemically synthesizing the fibronectin, which can compete with the original fibronectin of the extracellular matrix. The cells were found to bind with a specific region on the fibronectin that contains a three amino acid sequence Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation (Arg-Gly-Asp).

Value Addition: Do you Know? Heading Text: Figures showing the role of fibronectin in the migration of cells during frog gastrulation Body Text :

a) Interior of normal frog gastrula (ar = archenteron; bc=blastocoel)

b)

Exterior of normal frog gastrula (yp = yolk plug)

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The early development of Xenopus: Cleavage and Gastrulation

c) Interior of gastrula anti-fibronectin antibodies

injected

with

d) Exterior of gastrula injected with anti-fibronectin antibodies (ec = ectoderm; mes= mesoderm; end = endoderm)

Source: http://worms.zoology.wisc.edu/frogs/gastxen/gxmigra3.html

Boucaut et al injected large volumes of chemically synthesized small peptides containing those three amino acids into blastocoels of salamander embryos, just before the gastrulation begins. If the fibronectin is so essential for cell migration, then the cells that will bind to these soluble peptides should not migrate. This is because the soluble peptides do not provide the „pathways‟ on which the cells would normally migrate and thus stop the involution. The same result, as was hypothesized, was observed in the embryos undergoing gastrulation. They could not observe the migrating cells underneath the ectoderm in experimental embryos. Moreover, mesodermal precursor cells remained outside the embryos and formed a convoluted cell mass. The use of other synthetic peptides with Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation the embryo, did not derail the movement of cells and hence gastrulation in the embryo. The mesodermal cells adhere to the fibronectin molecule through the alpha vβ1 integrin protein. The use of antibodies against the fibronectin or a subunit of the integrin molecule, that is a part of the fibronectin receptor, can stop the migration of the mesodermal cells. The alpha v subunit of the integrin molecule appears on the surface of the mesodermal cells just prior to gastrulation and remains throughout the gastrulation and disappears from the cell surface as soon as gastrulation ends. Thus, the time of synthesis of the fibronectin receptor is an indication of the mesodermal cells to begin, continue or stop migration. The fibronectin containing extracellular matrix, provides for attachment of the mesodermal cells and also for the direction of the cell movement. Shi et al (1989) showed that if we place extra fibronectin lattices, the salamander IMZ cells will move in wrong direction and in the direction wherever they find the lattice. The convergent extension pushes the migrating cells upward towards the animal pole in Xenopus. The fibronectin fibrils are necessary for head mesodermal cells to flatten and they also extend broad processes in the direction of their migration. Delarue et al (1985) showed that in toads, certain inviable hybrids, the migration of the cells is arrested during gastrulation as they do not secrete the fibronectin fibrils. To summarize, the extracellular matrix of the blastocoel roof, and more specifically, its fibronectin component, is important for mesodermal migration during amphibian gastrulation. Click the link and see a morph of confocal optical sections illustrating the internal movements during gastrulation.: https://www.youtube.com/watch?feature=player_embedded&v=YlUsUH9G1Mo

Value addition: Interesting to know Heading text: Xenopus laevis stage series: complete Body text: Click on the link : http://www.xenbase.org/anatomy/alldev.do and check out the complete stage series of Xenopus laevis in digitized form based on Digitized images and developmental data from Nieuwkoop and Faber (1994) Normal Table of Xenopus laevis (Daudin). Garland Publishing Inc, New York ISBN 0-8153-1896-0. Source: http://www.xenbase.org/

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The early development of Xenopus: Cleavage and Gastrulation

SUMMARY 1. Although the amphibian cleavage is holoblastic, but it is unequal due to the presence of large amount of yolk in the vegetal hemisphere. 2. The general fate map of the fertilized amphibian egg is controlled by the transcription factor VegT and Vg1. 3. The three main events in amphibian gastrulation are: i) Invagination of bottle cells ii) Involution of mesoderm iii) Epiboly of the ectoderm 4. The direction of the involution is determined by the rotation of the cytoplasm at the vegetal hemisphere. 5. The bottle cells of Xenopus play important role in initiating the gastrulation, but they are not needed subsequently. 6. The involution of the IMZ cells is required for the formation of the archenteron. 7. The ectodermal epiboly and the convergent extension of the mesoderm are stimulated by intercalation events, which cause several tissue layers to merge. 8. The driving force for convergent extension is presence of two cell adhesion molecules: paraxial protocadherin and axial protocadherin. 9. The animal cap and noninvoluting marginal zone (NIMZ) cells expand by epiboly to cover up the entire embryo. 10. Fibronectin, present in the extracellular matrix, plays an important role in the migration of the mesodermal cells into the embryo. Click on the http://wiki.xenbase.org/xenwiki/index.php/Xenopus_development_ stages and check out the various images of Xenopus development stages

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The early development of Xenopus: Cleavage and Gastrulation

EXERCISE/PRACTICE Q.1 Define a) Epiboly b) Gastrulation c) Pharyngeal chordamesoderm Q.2 Differentiate between a) Involution and invagination b) Ectoderm and endoderm c) Animal hemisphere and vegetal hemisphere d) Micromeres and macromeres e) Blastocoel and archenteron f) IMZS and IMZD Q.3. Give contribution of the following scientists a) Kalt b) Nieuwkoop c) Gerhart et al d) Løvtrup e) Delarue et al Q.4. Fill in the blanks i) IMZ contains prospective ____________________________ of archenteron in its superficial layer. ii) An amphibian embryo containing 16 to 64 cells is commonly known as _________________ iii) The first cell adhesion molecule that keeps the amphibian blastomeres together is _________________________ iv) The fate map in amphibians is controlled by transcription factors _______________ and ___________ v) The induction by sperm is necessary for determining the ______________ of rotation in egg cytoplasm in amphibians. vi) The convergent extension is controlled by two cell adhesion molecules i.e._____________________ and _________________ . Q.5. Draw self explanatory diagram of an amphibian embryo after completion of gastrulation. Q.6. Multiple choice questions a) The NIMZ cells are found on i) Dorsal lip of blastopore ii) Ventral lip of blastopore iii) Lateral lip of blastopore iv) Surface of the embryo Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation b) The cleavage in amphibians is i) Holoblastic, complete iii) Radial, incomplete

ii) Holoblastic, incomplete iv) Meridional, complete

c) The so called „pathway‟ for migration of the mesodermal cells during involution is formed by i) CAMs ii) Myosin iii) Fibronectin iv) Actin d) The first cells to form slit-like blastopore in amphibian embryo are i) IMZ cells ii) Apical cells iii) Bottle cells iv) Endodermal cells Q.7. what are „bottle‟ cells? What is their significance in amphibian gastrulation? Q.8. Describe in detail the phenomenon of convergent extension.

GLOSSARY Animal hemisphere: The region of the fertilized egg which does not contain yolk. It is the region where the sperm penetrates the egg. Archenteron: the cavity formed by the involution and invagination of bottle cells and marginal zone cells. It obliterates the blastocoel. Blastocoel: The cavity present at the centre of the blastula Blastomeres: The cells present in a blastula. They can be of two types, as in case of amphibians, due to unequal cleavage and are called as micromeres and macromeres Blastula: A hollow ball of cells or blastomeres consisting of a cavity, called blastocoel, at the centre in early stages of embryonic development in animals. Ectoderm: The superficial layer surrounding the embryo after gastrulation and gives rise to nervous system and sense organs etc. Endoderm: The innermost layer lining the archenteron and gives rise to the digestive tract and its derivatives Epiboly: The thinning and spreading of the cell layers at the surface of the embryo. Gastrulation: The entire cascade of movements/migration of cells of blastula, such that they arrange themselves into prospective endoderm, ectoderm and mesoderm. These three layers further give rise to all the organs of the animal. Institute of Life Long Learning, University of Delhi

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The early development of Xenopus: Cleavage and Gastrulation Invagination: Movement of cells inward from the surface so that it forms an inpocketing Involuting Marginal Zone (IMZ): The marginal zone that involutes and forms the endoderm and mesoderm. Involution: A tissue sheet rolls inward such that it forms an underlying sheet. Macromeres: The blastomeres present in the vegetal hemisphere in amphibians. They undergo slower cleavage due to large amount of yolk. Marginal zone: The zone surrounding the equator of the blastula, where animal and vegetal hemisphere meet. Mesoderm: The layer present between the endoderm and ectoderm. It gives rise to muscles, bones, somites etc. Micromeres: The blastomeres present in the animal hemisphere in amphibians. They show faster cleavage. Noninvoluting Marginal Zone (NIMZ): The cells on the surface that expand and cover the embryo by epiboly. Vegetal hemisphere: The region opposite to the animal hemisphere and contains large amount of yolk.

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The early development of Xenopus: Cleavage and Gastrulation

REFERENCES 1. 2.

Developmental Biology: S.F. Gilbert http://www.ncbi.nlm.nih.gov/books/NBK26863/

SUGGESTED READINGS 1.

Molecular developmental biology: T Subramoniam

USEFUL WEB LINKS    

http://genome.wellcome.ac.uk/doc_WTD020799.html http://guides.library.harvard.edu/content.php?pid=237931&sid=2016930 http://www.xenbase.org/anatomy/intro.do http://worms.zoology.wisc.edu/frogs/gastxen/gxmigra3.html

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