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Handout Gastrulation CBT, 2009

This module is based on the following books :

'Principles of Developement', Lewis Wolpert, et al., Third edition, 2007Published by: Current Biology Ltd. and Oxford University Press.

'Developmental Biology', Scott F. Gilbert, Eighth edition, 2006Published by: Sinauer Associates, Inc. Publishers.

Introduction

Summary

This module is on the process of gastrulation.Gastrulation occurs during early embryonic development in animals.During this process endoderm and mesoderm become localized inside the embryo. "Gastrulation is the process of highly coordinated cells and tissue movement, whereby the cells

of the blastula are dramatically rearranged." (Gilbert) "Gastrulation is the process, whereby endoderm and mesoderm form the outside to the inside

of the embryo, giving rise to internal organs." (Wolpert)At the end of gastrulation:- mesoderm and endoderm are located inside the embryo.- ectoderm is completely covering the outside of the embryo.

Endoderm and mesoderm give rise to internal organs.

Objectives

After studying this module you should be able to:- describe the aims of gastrulation- describe the 5 different mechanisms of gastrulation- relate the 5 different mechanisms to examples in the animal kingdom- understand and recognize the 3-dimensional movements of cells during the gastrulation

processes- describe what tissue and organs are derivatives of the endoderm, mesoderm and ectoderm

What you already should know

- Early embryonic development, leading to blastula stages of Xenopus, sea urchin, zebrafish,Drosophilaand chicken.

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Gastrulation Mechanisms

Formation of mesoderm

Depending on the species, mesoderm is formed either before or during gastrulation.

Before gastrulation:For example in the frog (amphibia) the formation of the mesoderm starts around the 64-cellstage. It continues until the onset of gastrulation.

During gastrulation:In sea urchins (echinoderms), for example, the formation of mesoderm is part of thegastrulation process.

Mechanisms of gastrulation

There are 5 different kinds of gastrulation movements.

1) Epiboly: extension of a sheet of cells causing movement of this sheet overpresumptive deeper layers

2) Invagination: indentation of a region of cells resulting in the formation of a 'dimple'

3) Involution: inward movement of a layer of cells

4) Delamination: splitting of a tissue into 2 or more parallel cellular layers

5) Ingression: migration of individual cells from a surface layer to internal

One or more of these mechanisms can be responsible for gastrulation. This depends on thespecies.

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Convergent extension

In most species very extensive cell movements take place during gastrulation.Often cells 'crawl' in-between each other (intercalate) and elongate (extend).This process is called convergent extension.

Two types of convergent extension are known.1) Radial intercalation: A double (or multiple) layer of cells intercalate in such a way that a single

layer of cells is formed.As a result, the surface of the layer of cells is enlarged.

for example: ectoderm

2) Medio-lateral intercalation: Cells of a single layer intercalate medio-laterally. Due to thistype of intercalation, the cells form an elongated structure.

for example: endoderm,mesoderm, notochord

Echinoderms

Sea Urchin : early blastu la

Below you see an early blastula stage embryo (left) and its schematic drawing (right).

The embryo is a hollow sphere (blastula) with a large cavity, the blastocoel.The cells surrounding the cavity are connected to each other by tight-junctions. These cells forman epithelial layer, called the blastoderm.

All cells have cilia on their external surface. Because of ciliary movements the embryo can rotate.The external face of the cells is covered by the hyalin layer. This is a layer of extracellular matrixproteins.

At the blastocoelic side, the cells are covered with an extracellular layer. This layer is the basallamina.

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Sea Urchin : late blastula

After the formation of a blastula, the sea urchin embryo flattens andthickens at the vegetal side.This flattening is very well visible and is called the vegetal plate.

Pane 7 Question: Click on the vegetal plate in the figure on the right.

At the animal pole, some cells developed very long cilia. This group ofcilia is called the apical tuft.

Sea Urchin : fate map

A fate map illustrates what the different parts of the embryo will form later in(normal) development. This can be drawn at the late blastula stage.

The cells that will later form the mesoderm are all derivatives (descendents)

of the micromeres. Micromeres are small cells that result from unequalcleavage during early animal development.Remember that in sea urchin embryos the micromeres are located at thevegetal pole.

Sea Urchin: onset of gastrulation

In the sea urchin the gastrulation starts in the vegetal plate in individual cells.Some cells of the vegetal plate loose their affinity for their neighbouring cells and for the hyalinlayer. Moreover, their affinity for the basal lamina is increased. These cells change shape and formfilopodia.Finally, these cells leave their epithelial configuration and migrate into the blastocoel.

This process is called ingression.

After ingression, the cells that migrated into the blastocoel are called primary mesenchyme cells(PMC).In normal development about 64 PMC are formed. All PMC are derivatives of the micromeres.

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In animals in which the blastopore forms the anus, the mouth is formed secondarily. Theseanimals are called deuterostomes.In animals in which the blastopore forms the mouth, the anus is formed secondarily. Theseanimals are called protostomes.

Formation of the blastopore solely results from changes in cell shape due to apical constriction.

This is confirmed by computer simulations.

The hyalin layer consists of an inner and outer layer.The microvilli of the vegetal plate cells extend throughthe hyalin layer.

Their cytoplasm contains secretory vesicles with theenzyme chondroitin sulfate proteoglycan (CSPG).

At the onset of archenteron formation, the secretoryvesicles secrete CSPG into the inner lamina of thehyalin layer.The CSPG absorbs water and consequently theinner lamina swells. The outer lamina to which it isattached, does not swell. This causes bending of thehyalin layer and consequently the attached epithiliummoves further inward.

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Sea Urchin: formation of the archenteron 2

After invagination the archenteron starts to elongate. The endodermal cells lining the archenteronundergo convergent extension by medio-lateral intercalation. This elongates the archenteron tube.

Pane 21 Question: How does the process of convergent extension affect the diameter of thearchenteron tube?

Sea Urchin: formation of the archenteron 3

The onset of the final extension is characterized by yet another ingression of cells, the secondarymesenchyme cells (SMC). This ingression occurs at the top of the archenteron.The SMC remain on top of the archenteron. They form long filopodia with which they 'probe' theECM. The filopodia stick to one specific site at the animal side. Specificity for this site is based ondifferences in affinity to ECM proteins (differential adhesiveness).Once 'stuck' to this site the filopodia contract. This way the top of the archenteron is pulled up. Thearchenteron 'breaks through' at this site and forms the mouth.

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Amphibian

Xenopus: fertilization

The spermium enters at the animal half (hemisphere) of the oocyte. The point of entrance is calledthe 'sperm entry point' (SEP). The side where the SEP is located is the future ventral side of theanimal. The opposite side is the future dorsal side.

Pane 28 Question: Mark the dorsal and ventral side of the embryo.

The first cleavage is through the SEP and the future dorsal side. It divides the embryo in a left andright side. Gastrulation starts at the opposite side of the SEP, just below the Spemann Organizer.The Spemann Organizer is a signaling center on the dorsal side of the amphibian embryo. Signalsfrom this center can organize new antero-posterior and dorso-ventral axes.

Reread Wolpert, pages 110-113 to refresh your memory about:

- cortical reaction- cortical rotation

Reread Wolpert, pages 135, 167-169, for more information about the Spemann Organizer.

Xenopus: gastrulation

In Xenopusembryos, the presumptive endoderm is located in the vegetal hemisphere. These cellsare rich in yolk. Yolk is used as 'food' by the developing embryo.

Pane 29 Question: Drag the items to theircorrect position.

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The involution that started atthe dorsal side extendslaterally. Finally it starts atthe ventral side. This way acomplete ring of involutingcells is seen. Some

endodermal cells remainvisible.These cells are called theyolk plug.

Xenopus: gastrulation

The figure below shows a cross section of agastrula stage Xenopusembryo.

Pane 34 Question: Drag the following items to theircorrect position.1. ventral lip2. lateral lip3. dorsal lip4. archenteron5. endoderm

6. mesoderm7. ectoderm8. yolk plug9. blastocoel

The pictures below show the tissuemovements that take place duringgastrulation of Xenopus.They are, however, not in the correctsequence.

Pane 35 Question A: Drag the pictures to

the table below and put them in thecorrect order, starting from the beginningof gastrulation.

Question B: In which pictures is theblastocoel visible?

Question C: In which pictures is thearchenteron visible?

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The figure below shows a ventral view of the Xenopusembryo.

Pane 37 Question: Drag the following items to the correct position.

We've discussed Xenopusgastrulation.Pane 38 Question: Which three gastrulation processes are important in Xenopus?

1.

2.

3.

Fish

Zebrafish: early development

In the figure above you see the early cleavages of the zebrafish embryo.

The first cleavages occur every 15 min. These claevages are confined to the animal half of theembryo. In this way a mound of cells is formed that sit on top of a large yolk cell.

At the 10th cell cycle, the blastoderm can be divided into two layers of cells:- the outer enveloping cells and- the deep cells.

Until the 10th cell cycle, the blastomeres remain on top of the yolk celland divide synchronously.

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Zebrafish: onset of gastrulationAfter the 10th cell cycle, the division of cells become asynchronous and thecells begin to move.The first cell movement is the movement of cells over the yolk cell.

Pane 41 Question A: How is this process called?

Once cell movement has reached about 50% of the diameter of the embryo, athickening occurs at the entire margin of the blastoderm. This thickening iscalled the germ ring.

At the germ ring, the presumptive endodermal and mesodermal cells turninwards.

Pane 41 Question B: What is the name of this process?

The endodermal and mesodermal cells are derivatives of the deep layer.

Due to involution, the deep cells form two layers of cells:- an outer layer called epiblast,- an involuted layer called hypoblast.The envelope layer lays on top of the epiblast.

The epiblast forms the outer epithelium and the neural tissue. The hypoblast forms thechordamesoderm and the adjacent mesodermal structures.

Zebrafish: the embryonic shield

At the future dorsal side of the embryo a localized thickening isformed: the embryonic shield. This shield is functionallyequivalent to the dorsal involuting cells of the Xenopus embryo(the Spemann Organizer).

After involution, the hypoblast cells convergeanteriorly and dorsally and extend in anteriordirection.

Pane 43 Question: This type of cell movement is known as

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The hypoblast cells form the chordamesoderm, the primordium of the notochord.

Convergent extension can be shown by visualizingcells that express the gene no tail. No tailis

expressed in presumptive notochord cells. In thefigures you see no tail-expressing cells in twodifferent stages of gastrulation.

In the figure below, epiboly has reached about 50%of the diameter of the embryo.

Pane 46 Question: Enter the names of the specified structures.

We've discussed zebrafish gastrulation.

Pane 47 Question: Which two gastrulation mechanisms are important during zebrafishdevelopment?

1.

2.

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The figure below shows a lateral view of the Drosophilaembryo.Gastrulation begins when the presumptive mesoderm invaginates in the ventral region.First the ventral furrow is formed. Then an internalized tube is formed. Cells leave the tube andmigrate under the ectoderm. Later they form the mesodermal organs.

We've discussed Drosophilagastrulation.Pane 52 Question: Which two gastrulation mechanisms are important during Drosophiladevelopment?

1.

2.

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Chick

Chick: early cleavages

The early cleavages in the chick embryo create a blastodisc (= blastoderm) above the enormousyolk cell. As cleavages continue, the central cells of the blastodisc become separated from the

yolk cell by the subgerminal cavity.The cells above the subgerminal cavity appear clear and therefore this areais called the area pellucida (pellucidus = transparant). The cells at themargin of the area pellucida appear unclear and are called the area opaque(opacus = non-transparant).

Chick: onset of gastrulation

From the area pellucida individual cells move intothe subgerminal cavity.

Pane 55 Question: How is this gastrulationmechnism called?

They form small islets of cells called thepolyinvagination islands.

At the posterior margin of the blastoderm a groupof cells migrates into anterior direction. As theymigrate they incorporate the polyinvaginationislands. The resulting layer is called the primaryhypoblast.

Now the blastoderm consists of two layers:

- the epiblast, and- the primary hypoblast.

The primary hypoblast does not contribute to theembryo proper. It forms part of the externalmembranes (for example the yolk sac).

Chick: the primit ive streak

As the hypoblast moves anteriorly, some cells at the posterior side in the epiblast thicken. Thisthickened area is the (early) primitive streak.

At the place of thickening of the epiblast the following occurs:- ingression of mesodermal cells from the epiblast into the blastocoel, and- migration of cells from the lateral regions of the posterior epiblast towards the center.

The thickening narrows and moves anteriorly to form the definitive primitive streak.

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The primitive streak moves anteriorly (progression) until about 60-75% of the length of the areapellucida. It marks the anterior-posterior axis. At the anterior end of the primitive streak a regionalthickening is seen. This thickening is called Hensen's node.Having reached 60-75% of the length of the area pellucida, the primitive streak regresses towardthe posterior end.

Pane 58 Question: Drag the following items to their correct position.

Chick: cell movement in the primitive streak

During the progression of the primitive streak, cells migrate through:- Hensen's node, and- the primitive streak

Cells that move through Henson's node migrate anteriorly. They form the anlage of the foregut,head-mesoderm and the notochord.Cells that move through the primitive streak form the majority of the endoderm and mesoderm.

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The presumptive endodermal cells move deeper than the presumptive mesodermal cells. Theendodermal cells displace the hypoblast cells to the sides. They form the endoderm and most ofthe external membranes. The rest of the external membranes is formed by the hypoblast.

All cell movements through Hensen's node and the primitive streak are movements of individualcells, not of a sheet of cells like in Xenopus.

The primitive streak of chick is functionally equivalent to a structure in Xenopusand in zebrafish.In these structures (presumptive) mesodermal cells migrate into the blastocoel.

Pane 61 Question: Which structure in Xenopus and zebrafish are we referring to?

Xenopus:

Zebrafish:

Overview

Pane 62 Question: Which of the gastrulation processes are important during development of eachof the following animals?

delamination epiboly ingression invagination involution

sea urchin

Xenopus

zebra fish

Drosophila

chick

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