The early development of Xenopus: Embryonic induction and Organizers
Zoology Lesson: The early development of Xenopus – embryonic induction and organizers Lesson Developer: Dr Meena Yadav College/Department: Maitreyi College, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
TABLE OF CONTENTS INTRODUCTION AXIS FORMATION IN AMPHIBIANS: THE EXPERIMENTS First experiment by Spemann Second experiment by Spemann PRIMARY EMBRYONIC INDUCTION Transplantation experiments by Hans Spemann and Hilde Mangold AXIS FORMATION IN AMPHBIANS: THE MECHANISM
The Nieuwkoop center
The molecular mechanisms at the Nieuwkoop center
The functions of the organizer
The genes/molecules expressed/present in the organizer
The goosecoid gene
The proteins of organizer I
Noggin
Chordin and Nodal-related 3
Follistatin
Bone morphogenesis protein -4 (BMP-4)
The proteins of organizer II
Cerberus Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
Frzb and Dickkopf
Model given by Glink et al
TRANSFORMATION
OF
ECTODERMAL
CELLS
INTO
NEURAL PLATE CELLS Induction: Regional specificity Factors involved in anterior-posterior regionalization in the neural tissue Posteriorizing factors
eFGF
Retinoic acid
Wnt3a
The left-right axis Why Xnr-1 is expressed only on left side of the embryo? SUMMARY EXERCISE/PRACTICE GLOSSARY REFERENCES SUGGESTED READINGS USEFUL WEB LINKS
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The early development of Xenopus: Embryonic induction and Organizers
INTRODUCTION The body of most of the vertebrates has three major symmetries: o
Anterior-posterior axis
o
Dorsal-ventral axis
o
Left-right axis
The signals for formation of each of these symmetries come as early as fertilization. The act of fusion of sperm with the egg in amphibians, causes cytoplasmic or cortical movements that rearrange the contents of the egg. This rearrangement is the key to the establishment of different body axes. As the cleavage divisions start, the daughter cells receive unique cytoplasm containing the required signals for the formation of axes. One set of signals received gives way to the next set of signals in queue. So, although it may be visible from outside that the anterior-posterior axis forms first and so on, the molecular events are in the process to initiate the formation of other axes in line. Thus, the formation of axes is the result of a complex interplay at the molecular level in the embryos. In the current chapter, we will focus on these intricacies in the amphibian body axes formation during embryonic development.
AXIS FORMATION IN AMPHIBIANS: THE EXPERIMENTS The formation of axes in vertebrates is an intriguing phenomenon. The determinants of axes are not localized in blastomeres, like in Drosophila, but are instead scattered and arise as a result of cell-cell interactions between neighbouring cells. This is a progressive phenomenon and the events occur in a cascade, each previous event opening the doors for the next event. It is a highly regulated phenomenon. Such a phenomenon where the interactions between the cells lead to the next event of development is known as „induction‟. The concept of inductive interactions was given by Hans Spemann at the University of Freiburg, for which he got the Noble prize in 1935.
First experiment of Spemann In 1903, Spemann demonstrated that every blastomere in an early embryo in newts is capable of producing an entire embryo as they have identical nuclei. After fertilizing the egg, he allowed few cleavage divisions and then used a baby‟s hair to lariate the zygote in the plane of the first division. After this, he partially constricted the egg such that all the divisions were restricted to one side. After few divisions, probably after 16-cell stage, one nucleus escapes from the side where division is happening (nucleated side) Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers to the other side, where no division was occurring (non-nucleated side). The cleavage divisions also started on the previously non-nucleated side. As soon as the cleavage started on the other side, Spemann tightened the lasso, such that the two sides were completely isolated. On both sides, twin larvae developed, one being little older than the other (Figure 1).
Figure 1: The first experiment by Hans Spemann Source: Author The conclusion of this experiment: The nuclei of early embryo are identical and each is capable of giving rise to an entire organism.
Second experiment by Spemann: Spemann tied the lasso perpendicular to the plane of first cleavage. This separated the dorsal and ventral sides of the embryo, rather than the right and left side as in the first experiment. The division continued on both the dorsal and ventral sides, but only the Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers future dorsal side gave rise to a normal embryo. The other side produced a mass of tissue which was called by Spemann as ‘Bauchstück’ i.e. the belly piece. This tissue contained epidermal cells (i.e. ectoderm) along with blood and mesenchyme (i.e. mesoderm) and gut cells (i.e. endoderm) but no dorsal structures like neurons, notochord or somites (Figure 2).
Figure 2: The second experiment by Spemann Source: Author These two experiments showed that the plane of separation of the early embryonic cells has a huge influence on the fate of the dividing cells. This also indicates that the blastomeres are in constant touch with each other through the chemical substances including cytokines, which affect the cytoplasmic composition of the blastomeres and in turn their fate. The cytoplasm of the amphibian egg undergoes rearrangement due to its movements after fertilization. In some eggs, these cytoplasmic movements cause a „gray crescent‟ area to be formed in the zygote, opposite to the point of sperm entry.
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The early development of Xenopus: Embryonic induction and Organizers
VALUE ADDITION: Things you should know Heading: Dorsal-ventral patterning and neural induction in Xenopus embryo Body Text:
Source: http://www.hhmi.ucla.edu/derobertis/EDR_MS/chd_page/DeRobertis_ARCDB_Fig_2.jpg
The first cleavage division divides the gray crescent into two equal halves. These blastomeres, if separated, produce two complete and normal larvae. But, if the first cleavage division is not equal, may be due to a different plane of the cleavage, the cytoplasmic composition of the two blastomeres is different and the blastomeres that contain the gray crescent, can only develop into a complete larva. The gray crescent area, thus, contains some important clue for the development of the embryo. It was found that this area contains the signals for the fate map and will produce the cells that initiate the gastrulation. Thus, this area will form the cells of the dorsal lip of the blastopore. The cells of the dorsal lip of the blastopore are the first to invaginate and start the movements of the other cells and finally establishing the three major layers i.e. ectoderm, mesoderm and endoderm. Spemann also demonstrated, in 1980, that the cells constituting early gastrula are not committed but the cells of the late gastrula are committed. He transplanted a region of prospective epidermal cells from an early gastrula, into another early gastrula, at a Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers position where the neural tissue will be formed. The transplanted cells also gave rise to the neural tissue. If the prospective neural tissue from an early gastrula is transplanted in another early gastrula at a region that will form the belly region, then the transplanted tissue also gave rise to the epidermal tissue, instead of the neural tissue (Figure 3).
(A)
(B)
Transplantation in early gastrula
Transplantation in late gastrula
Figure 3: Transplantation experiments by Spemann Source: Author The cells of the early embryo are not committed and can develop into the type of cells surrounding it, if transplanted. Thus, they are said to exhibit conditional development or regulative development or dependent development. But, if cells from Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers late gastrulae are transplanted in an early gastrula, they develop into the types of cells that would have formed if they were present in the late gastrula, irrespective of the surrounding
cells.
This
is
known
as
autonomous
development
click
the
(also
called
independent or mosaic development). For
more
information
link:
http://www.hhmi.ucla.edu/derobertis/teaching/lecture_5.pdf
PRIMARY EMBRYONIC INDUCTION Transplantation experiments by Hans Spemann and Hilde Mangold (1924) Their experiments showed that of all the cells present in early gastrula, the fate of only one type of cells was fixed i.e. the cells forming the dorsal lip of the blastopore. These cells were derived from the cytoplasm of the gray crescent area. When they transplanted the dorsal lip tissue into the presumptive belly skin region of another gastrula, the tissue continued to develop as the dorsal lip and also initiated the gastrulation and embryo development in the neighboring region. Also, it formed two embryos, instead of one, as there were two dorsal lips – one of the recipient embryo and the other that was transplanted. Spemann and Mangold used embryos of two newt species that were differently colored i.e. Triturus taeniatus (dark pigmentation) and Triturus cristatus (non-pigmented). This made it easier for them to distinguish between the host and donor tissues. Experiment: The dorsal lip tissue of the early gastrula of T.taeniatus was removed and transplanted into the ventral epidermis region of the early T. cristatus gastrula. The donor dorsal lip tissue invaginated
and
started
gastrulation.
The
donor
tissue
continued
to
self-differentiate and formed the chordamesoderm (the notochord precursor) and other normal mesodermal structures. As the movement of the donor-derived mesodermal cells continued, the host cells started contributing to the formation of the organs in the new embryo. In the donor tissue derived embryo, each somite could be seen containing pigmented as well as non-pigmented tissue. The dorsal lip cells interacted with the host tissues to form a complete neural plate using host ectoderm. Thus, two embryos were formed face to face. These results of Spemann and Mangold were confirmed by designing experiments using nuclear markers (Figure 4).
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The early development of Xenopus: Embryonic induction and Organizers
Figure 4: Transplantation experiment of dorsal blastopore lip Source: Author
Value addition: Interesting to know Heading text: Spemann-Mangold organizer experiment Body text:
The Spemann-Mangold organizer experiment repeated in Xenopus laevis. (Top) control Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers swimming tadpole; (bottom-right) Spemann organizer graft at the same stage. In the bottom-left embryo the size of the graft can be visualized as a white patch (Spemann organizer from an albino donor embryo) at early gastrula (vegetal view). The dorsal lip of the blastopore may be seen as a thin crescent opposite the graft. Source: Edward M. De Robertis and Hiroki Kuroda. Dorsal-Ventral Patterning and Neural Induction in Xenopus Embryos. Annu Rev Cell Dev Biol. 2004; 20: 285–308. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2280069/figure/F1/
Free
for
educational purpose
INSERT THE VIDEO: Video 1. Spemann-Mangold experiment Movie 1: Reenactment of the Spemann-Mangold experiment: The movie opens with photographs of Hans Spemann and Hilde Mangold (circa 1924) [from International Journal of Developmental Biology: Special Issue, The Spemann-Mangold Organizer (E.M. De Robertis and J. Aréchaga, Eds.), 2001, volume 45.]. Eddy De Robertis at the dissection microscope. In one of two embryos, the dorsal blastopore lip, the organizer, is clearly visible as a crescent. A square of organizer tissue is excised, with the help of a tungsten needle and forceps, in a freehand operation. The organizer is pushed into the ventral side of a recipient gastrula. One hour after transplantation, the graft has healed in the host embryo. Two days later, a Siamese twin with two perfect body axes is seen swimming. The organizer graft induced complete central nervous system and mesodermal somites in tissues of the host that would otherwise have become dorsal tissue. Source: http://www.hhmi.ucla.edu/derobertis/movie/Spemann.avi Movie by Edward De Robertis and Hiroki Kuroda. The term “organizer” was given by Spemann for the dorsal lip cells and their derivatives. The organizer performed two important functions: i.
It induced the host ventral tissues to change their fates and form the dorsal mesodermal tissue (like somites) and a neural tube‟
ii.
It organized host and donor tissues for producing a secondary embryo with apparent anterior-posterior and dorsal-ventral axes.
He also proposed that during normal development, the organize cells arrange the dorsal ectoderm into neural tube and the flanking mesoderm is transformed into anterior-posterior body axis. The interaction between chordamesoderm and ectoderm is not sufficient to produce a complete embryo. In fact, it initiates a series of events that are sequentially inductive. Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers The phenomenon whereby one embryonic region interacts and stimulates the behavior of the second embryonic region is called induction. During the embryonic development, there are several inductions, but the key induction is when the progeny of dorsal lip cells induce the formation of dorsal axis and neural tube and this is known as primary embryonic induction.
AXIS FORMATION IN AMPHIBIANS: THE MECHANISM The Nieuwkoop center Pieter Nieuwkoop et al (1969, 1973 & 1977) explained how the dorsal lip of blastopore acquired its unique properties. They demonstrated that its properties were induced by the underlying vegetal cells (presumptive endoderm). In an experiment, he removed the equatorial cells (presumptive mesoderm) from the blastula and allowed the animal cap cells (presumptive ectoderm) and the vegetal cap cells (presumptive endoderm) to develop. None of the two could develop any mesodermal tissue. However, when he recombined the two caps, the animal cap cells were induced to form the mesodermal structures (Figure 5). The question now arises, whether the animal cap cells forming the dorsal mesoderm or the ventral mesoderm (i.e. the induction of polarity) are dependent on dorsal-ventral polarity of vegetal fragment (i.e. endoderm)?
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The early development of Xenopus: Embryonic induction and Organizers
Figure 5:
Experiment to show that signals for mesoderm
formation come from the vegetal hemisphere Source: Author It has been found that the ventral and lateral vegetal cells induce formation of ventral and intermediate mesoderm and dorsalmost vegetal cells form the dorsal mesoderm components, which have the properties of the organizer cells.
These dorsalmost
vegetal cells of the blastula which have the ability to induce organizer cells are known as Nieuwkoop center (Figure 6). The Nieuwkoop center was demonstrated in the 32-cell embryo by transplantation and recombination experiments. The transplantation experiments performed by Gerhart and Gimlich (1984) and Gimlich (1985 & 1986) were analogous to those performed by Spemann and Mangold, except that they used blastulae instead of gastrulae. When they transplanted the dorsalmost vegetal cells from one blastula to the other blastula at the same position i.e. ventral vegetal side, they observed the formation of two embryonic axes.
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The early development of Xenopus: Embryonic induction and Organizers
Figure 6: The amphibian blastula showing Nieuwkoop center Source: Author
Dale and Slack (1987) combined single vegetal blastomeres from a 32-cell Xenopus embryo and the uppermost cells of the animal cap, that were fluorescently labeled, of another embryo at the same stage. The donor dorsalmost vegetal cell induced the cells of the animal cap to become dorsal mesoderm. The rest of the vegetal cells induced the cells of the animal cap either to form the intermediate or the ventral mesodermal tissues. The Nieuwkoop center is the result of the cytoplasmic rotation that occurs after fertilization. As has been seen, if the cytoplasmic rotation is disturbed by any means including the UV light, the embryo will not form the dorsal-anterior structures like head or neural tube. If these UV treated embryos are transplanted with the dorsalmost vegetal blastomeres from a normal embryo at the 32-cell stage, these embryos can resume the normal development. If the eggs are mechanically rotated towards the end of the first cleavage division, such that the future ventral side is upward, then two Nieuwkoop centers are formed, leading to formation of two dorsal lips fo the blastopore and two embryonic axes. So, the specification of the dorsal-ventral axis can be traced back to the point of the sperm entry.
The molecular mechanisms at the Nieuwkoop center
In Xenopus, there is a gene called Xenopus Brachyury (Xbra) that is present in presumptive mesodermal cells. The endoderm induces the formation of mesoderm by Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers causing the presumptive mesodermal cells to express Xbra. The Xbra protein is a transcription factor that activates the genes which produce mesoderm-specific proteins. All the vegetal cells can induce the overlying marginal cells to become mesoderm, only the dorsalmost vegetal cells can induce the overlying dorsal-marginal cells to become organizer. The dorsalmost vegetal cells contain β-catenin which is a major candidate that forms the Nieuwkoop center (Figure 7).
Figure 7: Fate of marginal cells Source: Author
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The early development of Xenopus: Embryonic induction and Organizers
Figure 8: Localization of β-catenin during early and late cleavage divisions Source: Author β-catenin is a multifunctional protein and also acts as an anchor for the cell membrane cadherins or as a nuclear transcription factor.
In Xenopus embryos, during the
cytoplasmic rotation following fertilization, β-catenin begins to accumulate in the dorsal region of the egg and this accumulation continues at the dorsal side throughout the early cleavage. The accumulation of the β-catenin can be seen in the nuclei of the dorsal cells. The region where the β-catenin accumulates covers both the Nieuwkoop center and the organizer. However, during later cleavage division, the cells containing β-catenin are limited to Nieuwkoop center (Figure 8).
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The early development of Xenopus: Embryonic induction and Organizers
VALUE ADDITION Heading: Spemann organizers and self regulation in amphibian embryos Body Text:
Fig. Embryonic self-regulation. The entire early embryo constitutes a self-organizing morphogenetic field, in which cells communicate with each other over great distances. This is demonstrated by experiments such as the one shown here, in which a Xenopus embryo was cut in half at the blastula stage. If it is ensured that both halves contain part of the dorsal organizer region, two perfect identical twins are obtained (an intact sibling is shown at the top of the figure). Experiment by Edward De Robertis; photograph by Hiroki Kuroda.
Source: https://www.hhmi.org/sites/default/files/Our%20Scientists/Investigators/derobertis_fig1a_lg%5B 1%5D.gif
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The early development of Xenopus: Embryonic induction and Organizers If the β-catenin transcripts are inhibited by the use of antisense oligonucleotides, the embryo fails to develop the dorsal structures. Thus, the presence of β-catenin is important for the formation of the dorsal axis. Also, if exogenous β-catenin is injected into the ventral side of the embryo, it causes production of a secondary axis. Few important points: 1.
β-catenin is part of the Wnt signalling pathway and is negatively regulated by Glycogen synthase Kinase 3 (GSK-3).
2.
GSK-3 plays an important role in defining the dorsal-ventral axis by suppressing the markers for the dorsal surface.
3. 4.
If the activated GSK-3 is added to the egg, it blocks axis formation. If the endogenous GSK-3 is knocked out by adding a dominant negative protein in ventral cells of early embryo, it leads to formation of a second axis.
An important question now arises: how does the β-catenin become localized to prospective dorsal cells of the blastula? Labeling experiments suggest that initially β-catenin is synthesized by cells throughout the embryo, by utilizing the maternal transcripts. But, later on it is degraded by GSK-3 mediated phosphorylation, specifically in the ventral cells. So, here occurs a critical event: movement of an inhibitor of GSK-3 to the cytoplasmic region opposite to the point of sperm entry. This is the initial signal for the determination of the axis. One such inhibitor is Disheveled protein (Dsh). Dsh is a suppressor of the Wnt signaling pathway. In an unfertilized Xenopus egg, Dsh is found in the vegetal cortex. After fertilization Dsh is translocated to the dorsal side of the embryo using the microtubules. On the dorsal side of the embryo, β-catenin is stable because GSK-3 is not active and hence, can not degrade it. In the ventral region of the embryo, GSK-3 initiates the degradation of the β-catenin (Figure 9). β-catenin can associate with other transcription factors and improve or change their properties. For example, the Xenopus β-catenin can associate with ubiquitous transcription factor Tcf3 and define the dorsal axis. If, however, any mutation causes a loss of β-catenin binding site on Tcf3, the embryos will develop without dorsal axis.
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The early development of Xenopus: Embryonic induction and Organizers
Figure 9: Regulation of dorsal-ventral axis formation Source: Author It appears that the complex of Tcf3 and β-catenin is important for activating many genes that are critical for the formation of the dorsal-ventral axis. One gene which is activated by their complex is – siamois. This gene is expressed in the Nieuwkoop center, just after midblastula transition. If this gene is induced to be expressed in the ventral vegetal cells, a secondary axis forms from the former ventral side of the embryo. Also, if the initial cortical cytoplasmic rotation is prevented, the expression of siamois is stopped. The Tcf3 protein inhibits siamois transcription, if it binds to its promoter in the absence of β-catenin. However, if Tcf3/ β-catenin complex binds to the promoter of siamois, it gets activated. The Siamois protein is critical for the expression of the genes associated with the organizer. It binds to the promoter of the goosecoid gene and activates it. The Goosecoid protein is essential for activating numerous genes in the organizer. The Siamois protein alone is not sufficient to activate an organizer. The expression of the goosecoid gene is maximum when there is expression of another protein i.e. TGF-β, in the vegetal cells, along with the Siamois protein. The expression of these two proteins should be synergic (Figure 10).
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The early development of Xenopus: Embryonic induction and Organizers
Figure 10: Regulation of genes expressed for dorsal axis formation Source: Author The cortical rotation activates β-catenin which in turn activates the siamois in the dorsal region of the embryo. The translation of messages including a factor of TGF-β family, generates a protein that permits activation of goosecoid, primarily in the cells that will become the organizer. The TGF-β family protein in the Nieuwkoop center induces the cells in dorsal marginal zone above them to express some genes whose product are transcription factors, that bind to promoter of goosecoid gene and together with siamois gene activate the dorsal marginal zone. The potential candidates that activate the TGF-β factor are: Vg1, VegT and Nodal-related proteins. All these proteins are synthesized in the endoderm. Agius et al (2000) have demonstrated that all of these factors act together in a pathway and of all these, the critical proteins are Nodal-related factors. They repeated the Nieuwkoop animal-vegetal recombination experiments with a specific inhibitor of Nodal-related proteins. They found that induction of gastrulation by the vegetal cells failed to occur. They also found the expression of three Nodal-related proteins (Xnr1, Xnr2 & Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers Xnr4) during late blastula stage, in a dorsal to ventral gradient in the endoderm. This gradient is a result of activation of Xenopus Nodal-related gene expression by the synergistic action of VegT, Vg1 and β-catenin (Figure 11).
Figure 11: Regulation of mesoderm differentiation Source: Author Agius and his group present a model in which dorsally-located β-catenin and vegetally located VegT and Vg1 interact to create a gradient of the Nodal-related proteins across the endoderm. The specificity of mesodermal regions lies with these Nodal-related proteins, for example: i.
Regions with little or no Nodal-related proteins (ventral mesoderm)
ii.
Regions with some Nodal proteins (lateral mesoderm)
iii.
Region with large amount of Nodal protein (organizer)
The Nodal proteins activate the goosecoid gene and the use of its inhibitors prevents the activation of the gene.
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The early development of Xenopus: Embryonic induction and Organizers
The functions of the organizer The organizer progressively becomes dorsal mesoderm and migrates under the dorsal ectoderm, where it induces the formation of the central nervous system. The organizer tissue has following major properties: i.
It has the ability to become dorsal mesoderm (prechordal plate, chordamesoderm etc)
ii.
It induces the surrounding mesoderm to become lateral mesoderm (otherwise it would have formed ventral mesoderm)
iii.
it can dorsalize the ectoderm into neural ectoderm
iv.
it can initiate the movements of blastomeres for gastrulation
v.
it induces the neural plate to become neural tube
In vertebrates, in general, the formation of dorsal-ventral axis is followed by the formation of anterior-posterior axis. Firstly, the dorsal axis is established, the movement of involuting mesoderm establishes the anterior-posterior axis. The mesoderm that involutes first through the blastopore lip gives rise to the anterior structures and the mesoderm that migrates through the lateral and ventral lips of blastopore gives rise to posterior structures. The cells of the organizer contribute to the formation of the four cell types: o pharyngeal endoderm o
prechordal plate (head mesoderm)
o
dorsal mesoderm (mainly the notochord)
o
dorsal blastopore lip
The leading tissues of the organizer during involution are the pharyngeal endoderm and prechordal plate which induce the formation of the forebrain and midbrain. The dorsal mesoderm forms the hindbrain and trunk. The dorsal lip of the blastopore forms the dorsal mesoderm and finally forms the chordaneural hinge that induces the formation of the tip of the tail.
The genes/molecules expressed/present in the organizer The formation of the organizer, actually involves the expression of many genes. The protein products of the genes at the Nieuwkoop center activate the transcription factors in the mesodermal cells above them. These transcription factors then activate the genes fo the organizer. There are several organizer-specific transcription factors that have been identified (Table): Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
The goosecoid gene (in Nieuwkoop center): The area of expression of the
goosecoid mRNA is associated with organizer domain in normal as well as experimentally treated animals. The treatment of the embryo with lithium chloride increases the organizer
mesoderm throughout the marginal zone and along with it the expression of the goosecoid gene also expands. If the eggs are treated with UV light before the first cleavage division, the
induction of the axes i.e. dorsal-ventral and anterior-posterior, are highly compromised. If the full length goosecoid mRNA is injected into the two ventral blastomeres at
the 4-cell stage in Xenopus, the progeny of both the blastomeres to involute, undergo convolute extension and form the dorsal mesoderm and head endoderm of a secondary axis. Some labeling experiments have shown that in case of the above treatment, the
injected cells can recruit the neighboring cells into the dorsal axis. Thus, to summarize, the Nieuwkoop center, activates the goosecoid gene in the organizer tissue. The protein product of the goosecoid gene is a DNA-binding protein which serves the following functions: a. Activates certain genes that are involved in the migration of the cells of the dorsal lip of the blastopore (i.e. involution and convergent extension) b. Determines the dorsal mesodermal fates of the cells that express it c. It enables the goosecoid expressing cells to involve the neighboring cells in the formation of the dorsal axis. d. Goosecoid also activates the gene Xotx2, which is critical for formation of brain, anterior mesoderm and presumptive brain ectoderm. The proteins of the organizer I
Goosecoid protein is present in the nucleus where it activates the genes that produce soluble proteins involved in organizing dorsal-ventral and anterior-posterior axes. Evidences of soluble proteins from organizer I: o
Hans Holtfreter (1933): He placed the amphibian embryo in high salt solution and observed that the mesoderm does not invaginates, instead it evaginates. Due to this, it does not underlie the ectoderm, as in normal embryos. This ectoderm is not underlain by the notochord and also does not form the neural structures. Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
o
Saxén (1961), Toivonen et al (1975), Toivonen and Wartiovaara (1976): They took the tissue from the dorsal lip of the blastopore and placed it on one side of a thin filter, so thin that no processes could cross it. A competent gastrula ectodermal tissue was put on the other side. After several hours, they observed the appearance of neural structures from the ectodermal tissue. However, neural factors could not be identified at that time, it was only after a quarter of a century that we could actually identify the molecules involved.
Conclusions drawn about induction after recent experiments: o
The ectoderm is actually induced to become the epidermal tissue. The agents are
bone
morphogenesis
proteins
(BMPs).
The
signals from
the
neighboring cells cause this induction. o
The ectoderm that is not induced to form the epidermis, forms the nervous system. Thus, by default, the „fate‟ of the ectoderm is to form the neural tissues.
o
The organizer blocks the above said induction by secreting certain molecules that induce the ectoderm to become neural.
NOGGIN Smith and Harland (1992) isolated first soluble organizer molecule. They constructed a cDNA plasmid library from lithium chloride treated dorsalized gastrulae. They isolated RNAs synthesized from sets of these plasmids and injected in ventralized embryos (i.e. embryos having no neural tube), which were produced by irradiating early embryos with UV light. From the cDNA library, those sets of plasmids were isolated which were able to restore formation of dorsal axis in the embryos. Likewise, using the screening techniques, single-plasmid clones were isolated whose mRNAs could restore the dorsal axis in the embryos. One of these clones contained the noggin gene.
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The early development of Xenopus: Embryonic induction and Organizers
Figure 12: Experimental evidence to identify the noggin gene Source: Author
Although maternal noggin gene is present in the egg, but the newly synthesized noggin transcript was first observed to be localized in dorsal lip of the blastopore, by Smith and Harland (1992). Later this gene was also expressed in the notochord. If 1-cell, UV-irradiated embryo is injected with noggin mRNA, then the formation of the dorsal axis occurs and a complete embryo is formed (Figure 12). The Noggin protein is a secretory protein and has two important functions: o It induces dorsal ectoderm to form neural tissue o
It dorsalizes mesodermal cells, which otherwise would contribute to ventral Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers mesoderm. o
Noggin inhibits the binding of BMP2 and BMP4 to their receptors by binding with them.
Chordin and Nodal-related 3
Chordin was the second organizer protein to be discovered. It was isolated from the clones of cDNA whose mRNAs were present in dorsalized but not ventralized embryos. The clones were tested by injecting them into ventral blastomeres. One of the clones was capable of inducing formation of a secondary neural tube from the ventral blastomeres and was identified as chordin gene. The chordin mRNA was found to be localized in the dorsal lip of the blastopore and later in the dorsal mesoderm of the notochord. The role of Chordin is similar to that of Noggin i.e. binding with BMP4 and BMP2 and thereby preventing them to bind to their receptors. It has been found that in zebrafish having a loss of function mutation for chordin, there is a greatly reduced neural plate and an enlarged region of the ventral mesoderm. A third protein is Nodal-related 3 (Xnr-3) in the organizer region. It functions by preventing the binding of BMP4 to its receptors.
Follistatin
This protein of the organizer by discovered by Ali hemmati-Brivanlou and Douglas Melton (1992, 1994).
They were working on protein activin to show its role in
mesoderm induction. So, they constructed a dominant negative activin receptor and injected in Xenopus embryos. The ectoderm of these embryos began to express neural-specific proteins. It was observed that the activin receptor (which normally binds to structurally similar molecules like BMPs) bound an inhibitor of neurulation. When the function of the activin was blocked, all the ectoderm became neural. In 1994, Hemmati Brivanlou and Melton proposed a model called „default model of neurulation‟. According to this model: The organizer functions by producing inhibitors of whatever blocks the neurulation. This means the normal fate of the ectpodermal cells is to become a neuron, and it has to be induced to form any other type of cell. This model was supported by Grunz and Tacke (1989), Sato and Sargent (1989) and Godsave and Slack (1989).
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The early development of Xenopus: Embryonic induction and Organizers The naturally occurring protein follistatin binds to and inhibits activin, so it was hypothesized that it could be one of the factors secreted by the organizer. The presence of follistatin in the organizer was proved by in situ hybridization of follistatin mRNA in dorsal lip of the blastopore and notochord. So, in summary, there is a neural default state and an induced epidermal state. This concept is in contrast to the default model of neurulation. The question now arises, what proteins are inducing the formation of epidermis and if they are really being blocked by the signals from the organizer?
Bone Morphogenesis protein -4 (BMP-4)
The most significant inducer molecule is bone morphogenesis protein-4 (BMP-4). The relationship between BMP-4 and organizer is antagonistic.
If the mRNA of BMP-4 is injected in the Xenopus eggs, all the mesoderm of the embryo becomes ventrolateral mesoderm, and there is no involution at the dorsal lip of blastopore.
However, if a dominant negative BMP4 receptor is overexpressed, it results in formation of two dorsal axes.
Till the year 1996, results from several laboratories proved that Noggin, chordin and follistatin are secreted by the organizer and all of them prevented the binding of BMP to ectoderm and mesoderm near the organizer. The binding of BMP to ectodermal cells activates the expression of certain genes like msx1, which in turn induces the expression of genes specific to epidermis. In mesoderm, BMP4 activates genes such as Xvent1, which give ventral phenotype to the mesoderm. The doses of the BMP4 protein also show different actions: a. Low doses of BMP4 : activate muscle formation b. Intermediate dose : induces mesodermal cells to become kidney c. High doses of BMP4: activates the genes that induce the mesoderm to become blood cells The different doses of the BMP4 are produced in situ by the mixing of BMP4 (from ventral and lateral mesoderm) and BMP4 antagonists (from organizer).
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The early development of Xenopus: Embryonic induction and Organizers
VALUE ADDITION Heading: Neurulation in Axolotl (Ambystoma mexicanum) Body Text:
(a)
Embryo is about two and a half days old; the neural folds are moving towards the
midline. Eventually the neural folds will close over the neural plate and fuse, creating a closed neural tube which will give rise to the central nervous system. (b)
Neural folds are in contact in the middle (future anterior half of spinal cord); future
brain region (right) and posterior half of spinal cord (left)
(c)
Late neurulation: The neural folds are in contact with each other throughout the
entire length except at the future brain region (left) (d)
Late neurulation in cross-section: It shows fusing neural folds (top); the cells in
the neural tube are wedge-shaped and columnar while the remaining ectodermal cells are cuboidal; underlying the neural tube is the notochord (seen projecting here); a mass of paraxial mesoderm is lying directly to the lower right of the neural tube. Source: http://www.muhlenberg.edu/main/academics/biology/courses/bio240/amphibian.html Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
The summary is as follows:
Organizer
Secretes
Noggin, chordin and follistatin
+
_ BMP4
+
Ventral ectoderm (Epidermis)
Ventralizer of mesoderm (blood cells and connective tissue)
The proteins of organizer II
The concept has been that all the neural tissues are induced by the organizer to become forebrain, and notochord represents the most anterior part of the organizer. But the most anterior regions of the head and brain are not underlain by notochord, but by pharyngeal endoderm and head mesoderm (prechordal mesoderm). This is known as the endomesoderm and forms the leading edge of dorsal lip of the blastopore. Recent studies have shown that the endomesodermal cells induce the most anterior head structures by blocking the Wnt pathways and blocking the BMP4.
Cerberus
Nonorganizer mesoderm: That a member of Wnt growth and differentiation factors i.e. Xwnt8, inhibits neural induction was shown by Christian and Moon (1993). This protein was found to be synthesized throughout the marginal mesoderm except at the dorsal lip of the blastopore. Thus there were two anti-neuralizing proteins secreted by nonorganizer mesoderm i.e. BMP4 and Xwnt8. Organizer mesoderm: Bouwmeester et al (1996) demonstrated that a secreted protein from the organizer i.e. cerberus, induces the most anterior head structures to be formed. Unlike the other proteins by the organizer, cerberus induces the formation of the cement gland (the most anterior region of the tadpole), olfactory placodes and eyes. If the cerberus mRNA is injected into the ventral vegetal blastomeres at the 32-cell stage, ectopic heads are formed. The cell injected with the cerberus mRNA and its neighboring cells contribute to the formation of the head structures. The cerberus gene is expressed in the pharyngeal endomesoderm cells and the cerberus protein can bind to both BMPs and Xwnt8 and thus inhibit their function. Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
Figure 13: Summary of the molecules secreted by Organizers I & II Source: Author
Frzb and Dickkopf:
Two more proteins are synthesized by the involuting endomesoderm: Frzb (pronunciation: “frisbee”) and Dickkopf. Frzb is a small soluble form of Frizzled, Wnt receptor protein, which binds to soluble Wnt proteins. It is synthesized by the endomesoderm cells beneath the head. The presence of excess Frzb inhibits Wnt signaling and the embryos lack ventral posterior structures, making only head. The Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers Dickkopf (meaning in German: “big head”/”stubborn”) protein also interacts with the soluble Wnt proteins. If antibodies against the Dickkopf protein are injected into the blastocoel, the embryos tend to have small, deformed heads with no forebrain (Figure 13)
Model given by Glinka et al (1997)
They proposed a new model for embryonic induction. They demonstrated that the formation of the trunk structures may be induced by blocking BMP signaling from the notochord. But for formation of the head, both BMP signaling and Wnt signaling needs to be blocked. This blockage is done by signals from the endomesoderm (Figure 14).
Figure 14: Model given by Glinka et al for embryonic induction Source: Author
TRANSFORMATION
OF
ECTODERMAL
CELLS
INTO
NEURAL PLATE CELLS All of the ectoderm does not become epidermis, specifically the dorsal ectoderm. There are specific factors that prevent conversion of dorsal ectoderm into epidermis. Once this is done, certain other factors must be activated that induce it to become neural tissue. The vital protein involved is neurogenin. The transcription factors that are present in the ectoderm in the absence of BMP induce the expression of neurogenin Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers while the transcription factors present due to BMP signaling suppress the expression of neurogenin. Neurogenin is a transcription factor which activates a cascade of genes whose products are required for the expression of the neural phenotype. One important gene activated by neurogenin is the gene for NeuroD, which is a transcription factor that activates the genes involved in the production of neural specific proteins (structural). Noggin and Cerberus induce the expression of other transcription factors in the ectoderm like Xenopus brain factor-2 (XBF-2) and which in turn represses the epidermal genes. Due to action of all these factors, their activation or repression, the dorsal ectoderm is converted into neural plate tissue.
Induction: Regional specificity The regional specificity of the neural tissue is very important as it involves formation of the forebrain, hindbrain and spinocaudal regions. All these tissues must be organized in an anterior-to-posterior direction. The regions of the neural tissue are specified by the signals from the organizer itself. This type of induction was demonstrated by Otto Mangold (1933), husband of Hilde Mangold. The experiment is as follows: He isolated four successive regions of the archenteron roof of the late gastrula of newt embryos and transplanted them into the blastocoels of the early gastrula embryos.
Most anterior portion of the archenteron roof: induced formation of the balancers and parts of the oral apparatus.
The next part behind the most anterior portion: induced formation of the various head structures including eyes, nose, balancers and otic vesicles.
The third portion in line of the archenteron roof: induced the formation of the hind brain structures
Most posterior section of the archenteron roof: induced the formation of the dorsal trunk and tail mesoderm.
Some other experiments performed by Mangold (1933) are: a. If the dorsal blastopore lips from the early gastrulae of salamander are transplanted into early gastrulae of other salamanders, they form secondary heads. b. If the dorsal blastopore lips from the later gastrulae of salamander are transplanted into early gastrulae of other salamanders, they form secondary tails. The results of these experiments suggest that the first or initial cells of the organizer that enter the host embryo, induce the formation of the brains and heads while cells of the dorsal lip from the embryos at the later stage induce the cells above them to Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers become spinal cords and the tails.
Factors involved in the anterior-posterior regionalization in the neural tissue By the 1950‟s several experiments were done that provided the evidence that there are two gradients in the amphibian embryos: a. Dorsal gradient: having the ability to transform into neural tissue i.e. „neutralizing‟ activity. This activity comes from the organizer that induces the ectoderm to become neural tissue. b. Caudal gradient: having the ability to form posterior part of the embryo including mesodermal tissues i.e. „mesodermalizing‟ activity. This activity originates in the posterior of the embryo. Kolm and Sive (1977) have proposed that neutralizing and posteriorizing signaling systems work independently.
The neuralizing factors are:
chordin, noggin etc
The posteriorizing factors are:
eFGF, retinoic acid and Wnt3a
Posteriorizing factors
Embryonic Fibroblast Growth Factor (eFGF)
Fibroblast growth factors (FGFs) can transform anterior neural tissue into posterior neural tissue.
In an experiment, the early gastrula ectodermal tissue, which was not underlain by dorsal mesodermal tissue, was isolated and was neutralized by Noggin, Chordin or Follistatin. This led to the accumulation of the factors representative of the anterior-type neural markers in the region.
When the ectodermal tissue from the early gastrula was incubated with a neural inducer and FGF, the expression of the posterior neural markers was more. FGF-2 also induces the forebrain tissue to express genes involved in the hindbrain formation.
If the embryos are induced, in vivo, to express dominant negative FGF Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
receptors, the FGF signaling is blocked. This leads to the production of tadpoles that lack posterior segments. FGF-2 is not secreted by the embryo at the posterior side in Xenopus and it is also not localized in the embryo. However, a FGF molecule similar to the mammalian FGF-4 is found in Xenopus, it is called embryonic FGF (eFGF). It is found in the posterior and tail bud mesoderm of the Xenopus embryo and has same effects as FGF-2.
If the expression of the eFGF is more, it upregulates expression of the genes specific for the posterior region. One example of such overexpressed gene is Xenopus homologue of the caudal gene.
These posteriorly specific genes produce proteins that upregulate the expression of Hox genes that are involved in the anterior-posterior axis formation. Due to this, the posterior region of the embryo is specifically induced to form caudal nervous system.
The expression of the eFGF may be induced by the posterior notochord.
Retinoic acid:
Retinoic acid (RA) is a key factor in posteriorizing the neural tube in amphibians. If the neural tissue of Xenopus is treated with nanomolar and micromolar concentrations of retinoic acid, the development of the forebrain and midbrain is impaired and the impairment is directly proportional to the concentration of the RA used: i.
Lower concentration of RA: induction of neural tissue development is not inhibited as such, but fewer forebrain structures are produced. Retinoic acid affects the fate of both ectoderm and mesoderm.
ii.
If a gastrula is treated with retinoic acid, then its anterior dorsal mesoderm is not able to induce formation of head structures in the host embryo (Ruiz I Altaba and Jessell, 1991).
iii. If the anterior mesoderm of the untreated gastrula is transplanted in RA-treated gastrula, the host ectoderm was unable to respond to it. iv. It has been found that the dorsal mesoderm of the early Xenopus embryo contains a tenfold higher RA gradient in the posterior region than the anterior region. v.
Retinoic acid has been shown to activate the expression of more posterior Hox genes in amphibian embryos. Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
Wnt3a:
Xenopus Wnt3a is a caudalizing factor. In early neurula, it is found in neural ectoderm. If the neural ectoderm is isolated from the Xenopus gastrulae such that it remains connected to the dorsal lip of the blastopore, there exist neural markers in an anterior-posterior array. If the embryo is injected with Xwnt3a mRNA, causing overexpression of this protein, then the markers specific to the anterior region are lost. However, the relationship between the above three factors associated with the posterior patterning are yet to be established, although retinoic acid is involved specifically with hindbrain patterning, and eFGF with the patterning of the spinal cord. Wnt3a may be involved in suppressing the anterior genes while at the same time it allows the other two proteins to function normally.
VALUE ADDITION Heading: Embryos of Xenopus laevis inside the eggs. Body Text:
These are unique frogs as they lack tongue and visible ear Source: https://s-media-cache-ak0.pinimg.com/236x/dc/ff/43/dcff43bfa83fb0d4a85b6bd9b33c6d 20.jpg Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers
The left-right axis The tadpole larva looks symmetrical in the external morphology but it is not symmetrical in its anatomy as many internal organs, like heart and gut tube, are not evenly distributed. Thus, there exist three axes in the amphibian embryo: dorsal-ventral, anterior-posterior and left-right axes. In all vertebrates that have been studied for specification of their left-right axes, it has been found that it is under the control of the expression of a nodal gene, present in the lateral plate mesoderm on the left side of the embryo. In Xenopus, this gene is Xnr-1 (Xenopus nodal-related -1). If this gene is made to express on both sides of the embryo, the position of the heart and the gut tube becomes randomized.
Why Xnr-1 is expressed only on the left side of the embryo?
The answer lies in the events that occur soon after fertilization. The formation of the microtubules is an important event to occur for the cytoplasmic movements to happen that determine the fate of the embryo. If the formation of these microtubules is blocked, then there will not be any left-right axis formed. The Vg1 protein (expressed in the vegetal hemisphere) is converted into its active form primarily on the left side of the embryo. If we inject active Vg1 to the left side of the embryo, there is no significant effect on the development of the embryo. If however, it is injected to the right side of the embryo i.e. vegetal blastomeres on the right side, it leads to the expression of the Xnr-1 on both sides of the embryo and thus, randomization of the heart and gut tube location. If the active Vg1 is injected in a particular vegetal blastomere on the right-side, the entire left-right axis is inverted. In spite of the understanding of the correlation between these molecules/genes and the left-right axis, the exact mechanism that defines the left-right axis formation is still not known. The exact pathway of the signaling by Xnr-1 is not known but one of the crucial genes activated by Xnr-1 is pitx2. The gene pitx2 is normally expressed on the left side of the embryo due to its correlation with Xnr-1. If Pitx2 protein is injected on the right side of the embryo, the position of the heart and gut tube becomes randomized.
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The early development of Xenopus: Embryonic induction and Organizers
SUMMARY 1.
The organizer contains: pharyngeal endoderm, head mesoderm, notochord and dorsal blastopore lip.
2.
The organizer secretes proteins like chordin, Noggin and follistatin which block the BMP signaling and thus, prevent ventralization of the mesoderm and also inactivate the genes in the ectoderm that may induce it to form epidermis.
3.
In the anteriormost region, destined to form the head, the proteins Cerberus, Frzb and Dickkopf block the Wnt signal from the ventral and lateral mesoderm
4.
The first signal for involution comes from the organizer, which is induced by Nieuwkoop center, which is located in the dorsalmost vegetal cells.
5.
The Nieuwkoop center is identified by the presence of the protein Dishevelled which is translocated to the dorsal side of the egg due to cortical movements during fertilization.
6.
The Nieuwkoop center is formed by the accumulation of the β-catenin. The β-catenin is stabilized by the Dishevelled protein in the dorsal cells of the embryo.
7.
β-catenin associates with Tcf3 and makes a transcription factor complex that activates the expression of the siamois gene
8.
The siamois protein and TFG-β signal, may be Vg1, activate the goosecoid gene in the organizer.
9.
The goosecoid gene in turn activates other genes in the organizer and makes the organizer, specific for the involution initiation.
10.
The anterior-posterior specification of the neural tissue is under the control of posteriorizing signals like retinoic acid, eFGF and Wnt3a
11.
The left-right axis is specified by the signals as early as fertilization. One such signal is Vg1. By an ununderstood mechanism, this protein activates a Nodal protein entirely on the left side of the embryo.
12.
As in other vertebrates, the Nodal protein, in amphibians, activates expression of Pitx2, which is associated with the left-right axis formation.
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The early development of Xenopus: Embryonic induction and Organizers
EXERCISE/PRACTICE Q.1 Define i. “Neuralizing” activity
ii. Nieuwkoop center
Q.2 Differentiate between i. Mesoderm and endomesoderm ii. Organizer I and organizer II iii. Conditional development and autonomous development Q.3. Give contribution of the following scientists: i. Glinka et al
ii. Bouwmeester et al
iii. Hans Spemann
iv. Otto Mangold
Q.4. Give full form of the following: i. Dsh
ii. BMP
iii. eFGF
iv. GSK-3
Q.5. Fill in the blanks i. Neurogenin activates the gene _________________________ ii. BMP signaling is inhibited by __________________________ iii. The goosecoid gene is activated by ______________________ iv. Vg1 and nodal-related proteins are part of _____________ signaling pathway. v. β-catenin associates with _______________ to form a complex that acts as transcription
factor
and
activates
the
genes
for
______________________________________formation. vi. The siamois gene is expressed in the _____________________. Q.6.
Discuss
the
role
of
the
following
genes/factors
in
amphibian
embryogenesis: i. eFGF
ii. Noggin
iv. Wnt3a
v. Follistatin
iii. Xnr-1 vi. Xbra
Q.7. Multiple choice questions i. Neurogenin is a a. Carbohydrate
b. mRNA
c. Enzyme
d. Transcription factor
ii. The organizer is the name given to the a. Ventral mesoderm
b. Dorsal mesoderm
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The early development of Xenopus: Embryonic induction and Organizers c. Lateral mesoderm
d. chordamesoderm
iii. Which of the following is not an organizer protein? a. Chordin c.Frzb
b. Noggin d. Nodal-related protein
iv. The formation of cement gland, eyes and nasal placodes is induced by a. Noggin
b. Cerberus
c.Dickkopf
d. chordin
Q.8. Discuss, in brief, the functions of the organizer.
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The early development of Xenopus: Embryonic induction and Organizers
GLOSSARY Bone morphogenesis protein 4 (BMP4): Inhibits dorsal axis formation and ventralizes the embryo. It is inhibited by noggin, chordin, nodal-related proteins and follistatin Cerberus: It is a secretory protein from the organizer that induces most of the head structures to be formed. Chordin: Secretory protein of organizer I; inhibitor of BMP pathway and involved in dorsal-ventral axis formation. Dickkopf: Inhibitor of Wnt signalling; required for formation of anterior structures in the embryo. Disheveled (Dsh) protein: Present on the dorsal axis and is a suppressor of Wnt signalling pathway Embryonic fibroblast growth factor (eFGF): It induces the anterior neural tissue to form posterior neural structures. Follistatin: factor involved in dorsal-ventral axis formation Frzb: Found in the endomesoderm and inhibits Wnt pathway by binding to them; involved in formation of anterior structures goosecoid gene: Plays role in dorsal axis formation. Nieuwkoop centre: The dorsalmost vegetal cells of blastula that induce the organizer to start gastrulation Nodal-related proteins: Present in endodermal cells and inhibitors of BMP pathways. The three important ones are: Xnr1, Xnr2 and Xnr4. Noggin: Secretory protein of organizer I; involved in dorsal-ventral axis formation Organizer: The group of cells at the dorsal surface that initiate the involution of cells for the start of gastrulation Pitx2: The pitx2 gene is expressed on left side of the embryo and specifies the left-right axis, like position of the heart and gut tube Primary embryonic induction: Induction of the dorsal blastopore lip cells to form the dorsal axis and neural tube Retinoic acid: It posteriorizes the neural tissue in amphibians Siamois protein: Activates the expression of the goosecoid gene which in turn activates many genes in the organizer. Institute of Life Long Learning, University of Delhi
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The early development of Xenopus: Embryonic induction and Organizers Xwnt3a: It is a caudalizing factor and in early neurula, it is found in neural ectoderm. β-catenin: One of the factors that form Nieuwkoop centre. It can act as an anchor for cell membrane cadherins or as a transcription factor
REFERENCES
Developmental Biology: S.F. Gilbert
SUGGESTED READINGS
An introduction to embryology: B.I. Balinsky and B.C. Fabian
USEFUL WEB LINKS http://www.ncbi.nlm.nih.gov/books/ https://www.hhmi.org/research/cell-cell-communication-during-embryonic-induc tion
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