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Mesoderm Induction CBT, 2018 Hand-out CBT March 2018 Introduction

3. Books This module is based on the following books: - 'Principles of Developement', Lewis Wolpert, et al., fifth edition, 2015 - 'Developmental Biology', Scott F. Gilbert, eighth edition, 2006

4. Summary This module is on the process of mesoderm induction. Mesoderm is the third germ layer of the developing embryo. Mesoderm is important as it is present in all triploblastic animals. (Triploblastic animals are animals that have a three layered organization. These layers are ectoderm, endoderm and mesoderm.) It forms a large number of different organs and organ systems.

5. Aims and Objectives After studying this module you should be able to: - Understand the process of mesoderm induction in Xenopus - Know the latest model for mesoderm induction which is derived from relevant experiments - Tell about the different molecules that are involved in mesoderm induction - Tell something about the conservation of the mesoderm induction process in different animals Pre-requisites: You should know the early embryogenesis of Xenopus and Zebrafish including gastrulation.

6. Introduction Mesoderm is the third germ layer in the embryo. It forms a large number of different organs and organ systems such as skeleton, muscle, connective tissue, blood, the heart and the kidneys. Mesoderm is induced by an interaction of ectoderm and endoderm. Induction occurs during early embryogenesis. Depending on the species, mesoderm is either induced before gastrulation or during gastrulation. For example: - in Xenopus: mesoderm is formed before gastrulation - in Sea Urchin: mesoderm is formed during gastrulation

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Mesoderm induction

3. Q1A Dye experiment To construct a fate map the following experiments have been performed. At different stages individual cells of the embryo are injected with a fluorescent dye. This dye does not migrate between cells, and is therefore inherited by the progeny of the injected cell. After injection, the embryos are cultured until the tadpole stage. Which part of the tadpole will be labeled if the indicated cell is injected?

4. Q1B Dye experiment

Which part of the tadpole in this ventral view will be labeled if the indicated cell is injected?

5. Q1C Dye experiment 4 cell stage Which part of the tadpole will be labeled if the indicated cell is injected?

6. Explanation 4-cell experiment

7. Q1D Dye experiment 8-cell stage

Which part of the tadpole will be labeled if the indicated cell is injected?

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8. Fate map In order to find out which part of the embryo or adult is formed by which blastomere, cell lineage experiments can be performed. An overview of what each cell or part of an embryo will form during normal development is called a fate map.

9. Q2 Fate map construction Construct a fate map for the Xenopus embryo.

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10. Fate map: reality The experiments you just performed produce a theoretical outcome. In practice the cells move because of gastrulation movements. Therefore the progeny of the injected cells will be visible in a larger area than indicated in the tadpoles. This is an example of a real experiment. 1: the injected cell (C3) 2: cross sections of the developed tadpole with the progeny of C3 labeled (green/yellowish)

11. Specification map A specification map of an embryo shows how its blastomeres will develop when placed in a simple culture medium.

A specification map is stage dependent. This means that the specification of an embryo changes in time.

12. Fate versus specification A comparision of the fate and specification map of a blastula stage embryo reveals that they are not the same. It shows that during normal development some parts of the embryo form different tissue than if they were isolated at an early stage. This implies that certain tissues are determined by an inductive interaction. Fate map Xenopus blastula Specification map Xenopus blastula

Note that the specification map changes during development, the fate map stays the same. Specification maps of older embryos resemble the fate map more and more…

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13. Q3 Explants When isolated animal caps or vegetal parts of the Xenopus blastula are grown in culture medium no mesoderm is formed.

In an experiment animal caps and vegetal parts of the embryo are combined.

What happens if animal caps and vegetal parts of the embryo are combined?

14. Q4 Community effect The previous experiment shows that mesoderm is formed after an inductive interaction between the animal cap and the vegetal pole. In another experiment various numbers of animal cap cells are placed in-between two vegetal caps. Is every number of animal cells capable of forming mesoderm?

15. Q5 Inducer/recipient Mesoderm is induced by an interaction of ectoderm and endoderm. Which cells produce the inductive signal and which cells receive the inductive signal? The . . . . . . . . . . . . . . . . . cells produce the inductive signal, which is received by the . . . . . . . . . . . . . . . . . . cells

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16. Q6A Competence Whether the parts of the embryo are able to induce or receive the signal to form mesoderm is time and stage dependent. With this table you can determine the time window in which the vegetal and animal part are clearly able to induce or receive the signal for mesoderm induction.

What are these windows? Animal part: able to receive from . . . .to . . . . hrs after fertilization. Vegetal part: able to induce from . . . . to . . . . hrs after fertilization.

17. Q6A Competence The competence of animal and vegetal parts overlaps from 3.5 to 7 hours after fertilization. Consequently, what is the maximum time mesoderm induction can take place in the developing embryo?

18. Markers for mesoderm induction Cardiac actin mRNA and mRNA's of other muscle specific genes are used as markers for mesoderm induction. Muscle specific gene expression always starts 16 hours after fertilization. This is independent of when induction started, provided it took at least two hours.

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Sources of the inductive signal

3. Q1 Midblastula transition (MBT) The Midblastula Transition (MBT) in amphibian embryos is when:

• the embryos own genes begin to be transcribed (onset of the production of zygotic mRNA)

• cleavages become asynchronous • the cells of the blastula become mobile

The first cleavage takes 95 minutes, the following cleavages 35 minutes. Zygotic transcription begins after the 11th cleavage (halfway the 12th cell cycle). How many hours after fertilization is the embryonic transcription initiated?

4. Q2 Origin of signals for induction What does this reveal about the origin of the mRNA of signals for mesoderm induction?

5. Midblastula Transition (MBT) Zygotic transcription starts halfway the 12th cellcylcle around 8 hours after fertilization. During the first 11 cleavages all cells divide synchronously, with only an S and M phase. After the 11th cleavage the cell cycle also contains a G2 phase. Due to differences in the length of the G2 phase cleavages become asynchronous.

6. Q3 Cell movement Which process is driven by cell movement directly after MBT?

7. Overview of MBT in amphibian embryos Overview MBT in amphibian embryos: - the embryo's own genes begin to be transcribed - this gene expression is differential - the cell cycle elongates due to insertion of a G2 phase - cleavages become asynchronous due to differences in length of the G2 phase - the cells of the blastula become motile

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8. Xenopus development The movie is about early Xenopus development from one cell until MBT from an animal view. In this movie the first 12 cell divisions in the animal cap are visible. Link: https://youtu.be/GfBqRknZ2i8 In this experiment animal caps (tier A) are recombined with cells of the vegetal pole (D1 to D4). The results show that the kind of mesoderm induced depends on the vegetal pole cell used in the recombinant.

9. Q4A Mesoderm inducers Inductive signals must meet the following criteria: - mRNA must be maternally provided, since induction takes place before the zygotic genome is transcribed (before MBT) - the proteins must be present at a specific time - the proteins must be present at a specific place - the proteins must be able to induce mesoderm in animal cap cells Where is mRNA for mesoderm inducing agents localized in the early embryo?

10. Q4B Mesoderm inducers When is mRNA for mesoderm inducing agents translated?

11. Q5 Animal caps and vegetal cells In this experiment animal caps (tier A) are recombined with cells of the vegetal pole (D1 to D4). The results show that the kind of mesoderm induced depends on the vegetal pole cell used in the recombinant. In the table the ++ indicate the number of inductions (%). Which results belong to each recombination experiments?

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12. Vegetal hemisphere Different parts of the vegetal hemisphere induce different mesoderm: - Dorsal vegetal cells induce dorsal mesoderm (muscle and notochord). - Ventral vegetal cells induce ventral mesoderm (blood). This indicates different signals from the vegetal cells at the dorsal and ventral side. From these and other experiments a model for induction can be derived.

13. Four-signal model for mesoderm induction Two signals originate in the vegetal region: - Signal 1 in the ventral vegetal region induces ventral mesoderm at the ventral and lateral side. - Signal 2 in the dorsal vegetal region called the Nieuwkoop center. This dorsal mesoderm is

called the Spemann Organizer (O). Two signals originate in the marginal zone: - Signal 3 in the ventral mesoderm ventralizes the mesoderm. - Signal 4 in the Spemann Organizer dorsalizes the mesoderm by counteracting signal 3. As a

result intermediate mesoderm is formed. Signal 3 and 4 function later in development than 1 and 2.

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Search for inducers

3. Signals 1 and 2 Two groups of molecules have been investigated as candidates for signals 1 and 2 of the four-signal model of mesoderm induction: - TGFß-like molecules - FGF molecules You will study the TGFß-like molecules.

4. TGFß as signal 1 A strong argument for the involvement of a TGFß-like molecule comes from research on TGFß-receptors. TGFß-receptors work as dimers. After ligand binding a TGFß-receptor dimer is formed. Dimerization results in the activation of the intracellular kinase domain. The activated kinase domain phosphorylates downstream proteins leading to the activation of specific genes in the nucleus. To investigate the involvement of TGFß molecules in mesoderm induction a TGFß-receptor lacking the kinase domain was used.

5. Q1A TGFß as signal 1: experiment The artificial gene encoding this crippled TGFß-receptor was transcribed and its mRNA products are injected into a 1-cell stage embryo.

Which receptor combinations are able to form a dimer in the presence of TGFß molecules?

6. Q1B TGFß as signal 1: experiment Which of the receptor dimers are able to transmit the signal to the nucleus?

7. Q2A Results wildtype mRNA encoding either the wildtype TGFß-receptor or the mutant TGFß-receptor lacking the kinase domain is injected into a Xenopus two cell stage. What do you expect the resulting embryo will be if the wildtype receptor is injected?

8. Q2B Results wildtype What do you expect the resulting embryo will be if only the mutant receptor is injected?

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9. TGFß-like Vegetative 1 (Vg1) Vg1 is a potential mesoderm inducing agent since: - it is a TGFß-like molecule - its mRNA is present in the oocyte. Vg1 mRNA becomes localized to the vegetal side during oogenesis. The figure shows localization of Vg1 mRNA in the mature oocyte.

In situ hybridization with a fluorescent probe for maternal Vg-1 mRNA shows its localization (yellow) in the vegetal region of a mature amphibian oocyte.

Scale bar = 1 mm.

10. Vg1 and oogenesis Vg1 mRNA becomes localized to the vegetal part of the oocyte during stage V and VI of oogenesis. (Page 10 includes a movie as well.)

11. Q3A UV irradiation Imagine a 1-cell stage Xenopus embryo. During normal development, cortical rotation specifies the Nieuwkoop center in the dorsal vegetal region. The Nieuwkoop center is involved in the induction of the Spemann Organizer (signal 2). UV irradiation of the vegetal pole, well before the onset of the first cleavage (i.e. before about 80% of the first cell cycle) abolishes the cortical rotation by disrupting the microtubules responsible for movement of dorsalizing molecules. How does UV irradiation of the vegetal pole affect the formation of mesoderm?

12. Q3B UV irradiation UV irradiation of the vegetal pole, well before the onset of the first cleavage (i.e. before about 80% of the first cell cycle) abolishes the cortical rotation by disrupting the microtubules responsible for movement of dorsalizing molecules. By this UV irradiation the Nieuwkoop center formation is inhibited, and consequently no dorsal mesoderm and no Spemann Organizer is formed. The effect of UV treatment can be rescued by a high dose of Vg1. Which injection would rescue the effect of UV irradiation? Injection of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . of the oocyte

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13. Signal 2: induction of dorsal mesoderm (1) There is a clear difference in developmental potency between the dorsal and ventral mesoderm. During the induction process this difference must be created. When injected, TGF-ß (Vg1) induces ventral mesoderm at low concentrations and dorsal mesoderm at high concentrations. During normal development, TGF-ß is sufficient to induce ventral mesoderm. It is, however, not sufficient to induce dorsal mesoderm. This suggests that an additional signal is needed to induce dorsal mesoderm.

14. Signal 2: induction of dorsal mesoderm (2) The first suggestion that there is a dorsalizing signal, came from the following experiment (Spemann, 1938). Normally, the first cleavage is exactly through the sperm entrance point (SEP) and the grey crescent, separating the embryo into its future left and right half. Spemann succeeded, with experimental manipulation, to shift the cleavage plane 90 degrees.

After this he separated the two blastomeres of a two-cell stage embryo and grew the embryos until the tadpole stage. Some of them grew into normal embryos (left), but some were abnormal and contained only ventral tissue ('belly-piece') (left).

15. Q4 Spemann experiment Which halve(s) were abnormal?

16. Disheveled protein (Dsh) The Disheveled (Dsh) protein is involved in the induction of the dorsal mesoderm. Dsh inhibits the enzyme glycogen synthase kinase 3 (GSK3). When GSK is inactive, ß-catenin accumulates in the nucleus and acts as a transcription factor. ß-catenin is part of the Wnt signal transduction pathway and is a candidate for signal 2. GSK3 is present throughout the embryo. When active, GSK3 phosphorylates ß-catenin, initiating the degradation of ß-catenin. As a result no ß-catenin can enter the nucleus. (On page 16 two animations are shown: ‘With Dsh’ and ‘Without Dsh’)

17. Q5A Sperm entry During normal development, fertilization establishes the dorsoventral axis. (Note: this axis however is not fixed until shortly before the first cleavage, i.e. before about 80% of the first cell cycle.) This figure shows a cross section through an oocyte. In the oocyte the Disheveled protein (Dsh) is present in the cortex at the vegetal side. Indicate the side where the sperm will enter.

18. Q5B Dsh translocation To where are the vesicles containing the Dsh protein translocated during cortical rotation?

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19. Disheveled

During cortical rotation, the vesicles are translocated to the opposite side of the sperm entry point.

Disheveled is then released from the vesicles and is distributed into one-third of the dorsal side of the embryo.

Disheveled blocks the action of GSK3, thereby preventing the degradation of ß-catenin on the dorsal side of the embryo.

Consequently, at the dorsal side of the embryo ß-catenin accumulates in the nuclei and regulates expression of several genes that are important for dorsal mesoderm formation.

20. Q6 Experiment mutant GSK3 In order to show that inhibition of GSK3 activity induces dorsal mesoderm a number of different experiments can be performed. Predict the outcome of the following experiment. In one experiment, a dominant negative GSK3 enzyme was constructed. Note: this approach is similar to the one that was used to show that Tgf-ß receptors are involved in mesoderm induction. This was done by changing one amino acid in the ATP binding domain. The mutated GSK3 enzyme can bind ß-catenin but is unable to phosphorylate the molecule. An excess of mRNA encoding this mutated GSK3 enzyme was injected into the dorsal or the ventral part of the 2-cell stage embryo. What will be the result this injection? Injection Abormal (%)

Dorsal

Ventral

21. Experiment results

Pierce and Kimelman also injected dominant negative mRNA in the lateral side of embryos. The results were: Injection Normal (%) Double axis (%) Axis defects (%)

Dorsal >90 0 <10

Ventral 0 86 14

Lateral 20 10 70 S. B. Pierce and D. Kimelman, Development 121, 755-765 (1995) If the injection was more to the dorsal side then a normal embryo was formed. If the injection was more ventral then a double axis was formed. In other cases minor defects were found.

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22. Nieuwkoop center In the dorsal vegetal part of the fertilized egg the TGF-ß signal (Vg1) and ß-catenin overlap. This overlapping region is called the Nieuwkoop center. Here specific genes become activated by ß-catenin.

23. Q7 Model In the Nieuwkoop Center, ß-catenin together with the ubiquitous transcription factor Tcf 3, induces the expression of the siamois gene. The expression of siamois gives the Nieuwkoop Center the ability to induce the Spemann Organizer. Regulation of this gene can be summarized in a model. Link the regulations of the siamois gene to the correct arrows.

24. ß-catenin At the dorsal side ß-catenin and Tcf3 bind to the promoter of siamois, which is then transcribed. Siamois is a transcription factor of the homeobox type. It activates the expression of goosecoid when also a TGF-ß (such as Vg1) is active in that region. Goosecoid is a homeobox containing transcription factor. It is probably involved in the expression of other genes specific for the Spemann Organizer. (On this page an animation is shown of this process.)

25. Recap: Signal 1 and 2 What are the names of the molecules representing signal 1 and 2 of the four signal model of mesoderm induction? Signal 1: Signal 2:

26. Signals 3 and 4 In the previous chapter we discussed the induction of mesoderm in the marginal zone by the vegetal cells. The induced mesoderm consists of dorsal and ventral mesoderm. Dorsal mesoderm differs from ventral mesoderm because it expresses "dorsal” genes, including the gene goosecoid.

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But mesoderm is more than vegetal and dorsal mesoderm alone. There is also intermediate (or lateral) mesoderm that gives rise to a range of different organ(systems). The next question we want to answer is how different organs are generated in the lateral mesoderm. In the four-signal model this is the result of the signals 3 and 4.

27. Formation of the anterior-posterior axis The dorsal mesoderm invaginates during gastrulation. During invagination it induces the anterior-posterior axis in the embryo. Spemann and Mangold performed an experiment to prove that dorsal mesoderm is involved in the formation of the anterior-posterior axis in the embryo. They transplanted the dorsal blastopore lip, that consists of dorsal mesoderm, into the future ventral region of a recipient. The recipient was then allowed to develop.

28. Q8 Experiment Spemann and Mangold What will happen in the constructed embryo?

29. Candidates for signal 3 and 4 Two categories of proteins are involved in mesoderm patterning along the dorso-ventral axis:

1. Ventralizing agents (BMP-4, Xwnt-8) 2. Dorsalizing agents (Noggin, Chordin, Frizbee,

Follistatin) The distribution of these proteins corresponds to these catagories. The dorsalizing agents inhibit action of the ventalizing factors: - Binding of Nogging, Chordin and Follistatin to BMP-4 prevents BMP-4 from binding to its

receptor. - Binding of Frisbee to Xwnt-8 prevents Xwnt-8 from binding to its receptor Note: BMP-4 is expressed throughout the embryo. Xwnt-8 is only present in the marginal zone. Expression of noggin, chordin and frizbee is restricted to the Spemann Organizer.

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Other vertebrates

3. Introduction Until now we have looked at mesoderm induction in Xenopus only. But how about mesoderm induction in other vertebrates? Is there any conservation of the processes that induce mesoderm? Can we do similar experiments as in Xenopus, for example in birds?

4. Conservation in birds Part of a quail embryo is transplanted into a chick recipient. You expect that transplantation of a certain part of the quail embryo will result in the formation of a secondary axis in the chicken embryo. Which region of the quail embryo must be transplanted?

5. Conservation in zebrafish A similar experiment can be done in zebrafish. The embryonic shield of the zebrafish resembles the dorsal blastopore lip of Xenopus. Thus, in the zebrafish the embryonic shield functions as the organizer. Transplantation of a donor embryonic shield to the ventral side of a host embryo results two embryonic axes joined to the host's yolk cell.

6. Inducers in zebrafish The mechanism that induces the organizer seems to be similar in Xenopus and zebrafish. In both species ß-catenin localizes in nuclei of cells at the dorsal side of the embryo.

Nuclear localization of ß-catenin marks the dorsal side of the Xenopus blastula (large one). In the zebrafish late blastula (small embryo on the left), nuclear localization of ß-catenin is seen in the yolk syncytial layer nuclei beneath the furture embryonic shield. The brown points are nuclei in which ß-catenin is localized.

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7. Conservation in mouse A number of genes are expressed both in the organizer of Xenopus and in the organizer of the mouse embryo.

8. Conservation between vertebrates and invertebrates The induction of mesoderm and the formation of the anterior-posterior axis is conserved in vertebrates. In invertebrates similar genes are found. Until now, these genes seem to play no role in the induction of mesoderm in invertebrates. One of the animals that is well studied in this respect is Drosophila. None of the genes or mechanisms that are relevant in vertebrates is active during the process of mesoderm induction in Drosophila.