The aim of the assignment is to learn more about the development of the embryo using the CAL with particular focus on the development of the eye. This will include the key events in the first eight weeks post-fertilization, an overview of the important processes which occur and subsequently the formation of the primitive eye
For this assignment a CAL on embryology was shown which introduced the subject of the development of the embryo with particular emphasis on the development of the eye. Embryology is the study of the development of the zygote from fertilization to 8 weeks. After 8 weeks the embryo is known as a foetus and the external structure of the human form is clear to see; internally, organogenesis continues until birth and beyond.
Research is carried out on embryos of many different species of animals; the same events occur at a tissue level via the same processes, the differences between species is due to the differences in genotype and therefore the proteins produced. Mice and chicks are commonly used during research as they can be easily reproduced and the embryonic development is very similar to humans. The pictures displayed in the results section are taken from scanning electron micrographs of mice eyes1.
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The development of the embryo can be divided into stages and two widely used methods of doing so are the Carnegie stages and the 'post-ovulatory' stages. There are 23 Carnegie stages and these are used to describe the stage of maturity of a vertebrate embryo independent of its size or age. The postovulatory stage is used to describe the age of an embryo based on the last ovulation. Knowing the stage of maturity of the embryo or foetus at each day allows a developmental timeline to be produced and knowledge of the critical development stages at which major or minor anomalies occur.
After fertilization, the zygote divides and forms a 16 cell blastula; the next stage is gastrulation, the development of the three germ layers from which all cell types will originate; then neurulation, the first formation of the central nervous system. The essay will only include an overview of these processes as the available detail at both tissue and cellular level is vast. During the first eight weeks of life a miniature eye is formed, albeit without too much differentiation, and this will also be discussed.
In mammals, fertilization begins with the fusion of a sperm cell and an ovum (Fig 1) The egg has a thick glycoprotein coating, the zona pellucida, which is secreted by follicular cells, to which the sperm binds and releases digestive enzymes to penetrate the plasma membrane which activates the ovum to metabolise and begin the
process of mitosis. The acrosome reaction is activated when the sperm binds to the ZP3 protein and subsequently the fertillin protein in the sperm binds to integrins on the oocyte surface. This triggers a pathway which opens calcium channels in the sperm and across the egg; cortisol granules released by exocytosis and additional proteases prevent sperm from binding further (Slack et al 2001). Calmodulin may be active in regulating the calcium channels in the sperm (López-González et al 2001 ).
Fig 1 sperm and ovum Acrosome-reacted sperm bind to a second protein, ZP3, in the zona pellucida whereas non acrosome-reacted sperm will not - this is one mechanism to prevent polyspermy.
When the sperm enters the plasma membrane both cells begin DNA replication and a mitotic division. When the nuclear envelopes break down the chromosomes align on the mitotic spindle during pro-metaphase and the diploid number of chromosomes is restored in the daughter cell.
Inner cell mass
Fig. 2 The blastocyst The fertilized ovum is now known as the zygote, located in the oviduct, and moves through the oviduct to the uterus. The zona pellucida protects the zygote from attaching to the oviduct during this time and when the uterus is reached, the blastocoel, which has expanded within the zona pellucida, is able to hatch and bind itself to the uterine wall. During the first 2-3 days, cleavage of the zygote divides the cell at approximately one division every 12-24 hours. When the 8-cell stage is reached, compaction occurs which is where the cells cluster together to form a tight ball and tight junctions form on the outside. The next division brings the 16 cell zygote, the morula, and subsequently the blastocyst which has two separate areas. (Fig 2, right)
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The group of external cells develop into the trophoblast; this will attach to the endometrium of the uterus which stimulates the uterine mucosa to proliferate. The trophoblast cells have integrins which bind to proteins in the endometrium and secrete proteases to digest the extracellular matrix so that it remains implanted. The chorion, the fetal placenta, is formed from trophoblast cells and, together with high progesterone levels, this induces the formation of the maternal placenta, the decidua, so that nutrients can be obtained from the mother's blood supply (Slack 2001).
The internal morula is known as the blastocoel and increases in size due to the development of sodium pumps in the trophoblast which bring in water by osmosis and contains the inner cell mass (ICM) which will develop into the embryo. The ICM contains the pluripotent embryonic stem cells.
Gastrulation takes place between 14 and 21 days post ovulatory days and is the process of the formation of the three germ layers in preparation for the pending neurulation. The inner cell mass, now known as the gastrula, develops into the hypoblast layer and the epiblast layer, this latter layer will form the embryonic tissues. A structure known as the primitive streak becomes an axis of symmetry and the anterior- posterior ends can now be defined and are controlled by varying concentrations of different Hox genes (Gilbert 2000). The main event of gastrulation is the ingression of epiblast cells through the primitive streak and the creation of the ectoderm, mesoderm and endoderm layers2. The hypoblast cells combine with mesodermal cells to create the yolk sac and amnion which protects the embryo and foetus. The ectoderm, the outermost layer, will form the skin cells, brain neurons and parts of the eye; the mesoderm layer will differentiate into a variety of different cells including cardiac cells, muscle cells and erythrocytes; whilst the endoderm cells will develop into the lungs, thyroid and pancreatic cells.
Fig 3 Neural foldsNeurulation is the next development process and concurs with gastrulation. It includes the formation of the central nervous system and the first event is the development of the notochord derived from the mesoderm layer and the primitive streak. This signals the ectoderm layer above to form the neural plate with an indentation in the centre, the neural groove, which creates a right-left axis, occurring in Carnegie stage 8. Cell proliferation causes the edges of the plate to thicken and move upwards, with the neural groove in the centre, to become neural folds (Fig 3) and these gradually fuse together. They continue to displace themselves downwards away from the ectoderm until they fuse to form the neural tube which extends throughout the embryo.
Neurulation and gastrulation are not synchronized along the axis of the mammalian embryo with the anterior end developing faster than the posterior end. The two opens ends of the tube are called neurophores and neurulation is complete when all of the neural folds have adhered at the dorsal midline and this is occurs at 28 post ovulatory days. The neural tube is separated into neurons and glial cells with the brain and spinal regions separating from one another. The definitive separation of the two areas is achieved by pressure being exerted on the neural tube walls by the internal fluid which constricts the tissue at the top of the presumptive spinal cord. At the anterior end the brain develops into the prosencephalon, mesencephalon and rhombencephalon which form the primitive forebrain, midbrain and hindbrain respectively. Neural crest cells are located between the ectoderm layer and neural tube. These cells move into the periphery and this is aided by proteins which disassociate the tight junctions and also a reduction in cadherins which bind the cells; they are essentially moving from being epithelial cells to mesenchyme (Gilbert 2000). The neural crest cells differentiate into different cell types, aided by the Sox transcription factors (Hong and Saint- Jeannet 2005), such as neurons, melanocytes and connective tissue, as well as brain and spinal epithelia and glial cells and PNS cell and it is generally known that their residual location after migration will confirm which cell type they become.
Meanwhile, groups of mesodermal cells, known as somites, position themselves along the neural tube and will develop into vertebrae and skeletal muscle and dermis.
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The Development of the Eye
Neural plateAs previously mentioned, by 22 postovulatory days three primary vesicles will have developed at the anterior end of the embryo forming the primitive brain. Eye development begins when a pair of optic vesicles appears in the forebrain (Fig.3); they are connected to an optic stalk and both structures are made from epithelial cells. They continue to grow by cell proliferation and the signal to stop is when close proximity to the upper surface ectoderm is reached. It is understood that the induction
Fig. 4 Neural Plate and eye fields
Fig. 5 The lens placode lies in front of the optic vesicle
of the lens formation is due to the presence of the optic vesicle but research by Saha et al (1989) has proposed that previous mechanisms during gastrulation involving the ectoderm and early neural signals make the lens region biased to forming the placode and the optic vesicle proximity is a late factor. Both the optic vesicle and surface ectoderm thicken and the latter forms the lens placode. Then both surfaces invaginate inwards to produce the lens vesicle and the optic cup (Fig 5). This occurs quickly, taking about twenty four hours, due to increased mitosis and elongation of cells. The optic cup axis is important for the correct development of the neural retina and RPE.
The cell proliferation in the optic cup is not symmetrical as the ventral side grows more slowly than the dorsal side and this creates the choroidal fissure, a depression in the front part of the optic cup. The hyaloid artery enters the optic stalk and supplies the lens; this will later become the central retinal artery when the choroidal fissure closes. The latter occurs at around six weeks by fusion of the two edges so that the optic cup has two defined layers (outer and inner). The closure of the choroidal fissure occurs just after the lens forms by separating itself from the surface ectoderm to become an entity on its own.
Fig 6. Cross section of lens fibres The lens begins as a hollow ring of epithelial cells, which are now the only source of all lens cells, and an acellular capsule. The hollow is filled by the growth of primary fibres (Fig.6) in the posterior-anterior direction and these then lose their ability to proliferate and lose their DNA and organelles. The cells in the anterior lens epithelium are like stem cells as they continue to proliferate and are immortal. The daughter cells are lens fibres which elongate in the posterior direction to form Y shaped sutures. The production of lens fibres by the anterior epithelium does not stop and the new fibres are continually added to the lens throughout life (Oyster 1999)
By now, there are structures formed which are the precursors to many of the eye's components. From the outside in, the surface ectoderm develops into the corneal epithelium and the neural crest cells underneath become the corneal stroma and endothelium. The outer layer of the optic cup becomes the epithelial layers of the iris and ciliary body anteriorly and the RPE layer posteriorly whilst the optic stalk is the future
Fig. 7 Retina and RPE layers
optic nerve. The inner optic cup develops into the neural retina; by week 8 this can be seen as the thicker of two layers, the other being the RPE, both divided by a small subretinal space (Fig.7) In the embryonic stages, most of these cells have not differentiated and are still mesenchyme or epithelial cells; two exceptions to this are the lens cells and the pigment cells of the RPE which synthesise melanin by week 5. The differentiation of the retinal cells does not occur until around 12 weeks. Figure 8 shows the space between the lens and the cornea. As the primary fibres develop this space decreases and during week 7 the anterior chamber develops (Forrester et al 2002)
Fig. 8 Anterior chamber develops in the lens cavity
Primary vitreous has also formed, beginning before the choroidal fissure closed, with the formation of fibroblasts at the posterior optic cup at 5 weeks. Around 7 weeks the hyaloid artery supplying the lens gives off small capillary branches around which cells and collagen accumulate. The primary vitreous is later replaced by secondary vitreous again originating posteriorly and expanding forward.
Mesenchyme cells form a layer around the optic cup whereby its inner and outer layers will form the choroid and sclera respectively. The sclera, extraocular muscles and blood vessels are derived from the mesoderm. At the end of the embryonic period the optic nerve begins to develop with ganglion cells growing from the inner retina towards the optic stalk.
There are four important processes in the development of an embryo, akin to all animal species: differentiation, apoptosis, motility and growth. These processes all occur during and throughout the life of the embryo, foetus and neonate and into adulthood and are controlled by the expression of genes, growth factors and transcription factors. In embryology, it is particularly important to co-ordinate the growth of each tissue in the correct location for example the presence of Pax6, among other proteins, induces the surface ectoderm to invaginate when the optic vesicle is in close proximity (Li et al 1994). Transcription factors are groups of proteins which control the output of particular genes; many are known and grouped into families of similar structure, for example, the Pax family of transcription factors controls part of eye development and nervous system development. Pax6 (paired-box) is a transcription factor which is very influential in the development of the eye. It is expressed in both the lens placode epithelium and the anterior neuroepithelium and is thought to influence groups of cells to differentiate into their respective cell types and maintains cell proliferation (Forrester et al 2002) If Pax6 is missing then the eye may not develop at all. Pax2 is involved with the closure of the choroidal fissure. The absence of Pax6 can be the reason for the majority of cases of aniridia but it is not always the case for example recent clinical examination of 4 patients with aniridia by Traboulsi et al (2008) found Pax6 was fully functional and suggested this was the case for 20% of cases with more research needed. There are four main families of growth factors and these are used for paracrine signalling i.e. local, and these are thought to be important during induction. Induction is used to describe the process of the behaviour of cells in the presence of a specific cellular environment and is used for co-ordination and the functioning of the complex anatomical system. The hedgehog proteins are one example of a family of paracrine factors and the Sonic hedgehog is particularly relevant to the development of the central nervous system as it is produced by the notochord and, amongst other functions, ensures the correct placing of the sensory and motor neurons and contributes to retinal cell proliferation under the control of Vsx2 (Sigulinsky et al 2008).
The organisation and shape of the tissues and organs formed is the process of morphogenesis and is very complex, much is still unknown. In the embyro there are two main cells types: epithelial and mesenchyme. Epithelial cell sheets are held together by adhesion molecules, examples of which are cadherins. Cadherins attach to each other on adjacent cells and combine with catenins, which bind to the actin skeleton to form adherens junctions, and this combines the epithelial cells. Different cadherins have varying functions e.g. P- cadherins help to attach the trophoblast cells to the uterus. Cadherin binding is possible due to homophilic binding which separates the molecules into their respective groups and therefore will keep different tissues apart. Cadherins can also be inducers for example N-cadherin expressed in the neural plate is involved with early neurulation (Dietrich et al 1990). Throughout the development, motility of cells is undertaken by mesenchymal cells, derived from the mesoderm, which make up loose connective tissue; each organ has an epithelial and
The development of the embryo and indeed any anatomical system is always highly co-ordinated and complex and depends on all of the signals being sent at the correct time to the correct location. Obviously this is not always achieved and malformations may occur. With relevance to the development of the eye, the incomplete closure of the choroidal fissure can result in malformations in the uvea and the retina, known as colobomas. Some have a more detrimental effect than others, for example, if the RPE was affected then induction may not have occurred properly with the result of an absence of Bruch's membrane and the choroid so that the sclera is visible underneath (fig 9).
Fig.9 Coloboma of the RPE
Deformities also occur if the neural tube and optic vesicle fail to develop properly such as the absence of the eye or the presence of a dwarf eye which failed to develop, microphthalmos, but these are rare. Failure of the lens induction results in aphakia. The neural tube is the primitive spinal cord and brain. At the end of four weeks, neurulation is complete, its main purpose being the beginning of the formation of the CNS and also the origin of neural crest cells. If the neural tube does not close properly then spinal disorders may occur. For example, if the posterior end of the neural tube doesn't close then spina bifida may develop, if the anterior end does not close this could be fatal for the embryo as the brain will not be protected leading to anencephaly.
The testing of human embryos is particularly topical at the moment. The cells of the zygote are totipotent i.e. they can become any type of differentiated cell including the extraembryonic tissues; the latter discriminates them from pluripotent cells which can form any cell but cannot form a full organism. The process of differentiation switches on pathways in the cell which enable it to produce specific proteins and become a specialist cell after which it cannot turn back into the original pluripotent cell and is the case for all of its daughter cells. Stem cells are extremely useful as they are pluripotent. The study of human embryos has taken place for some years by using spare embryo's or unfertilized eggs from donors having IVF treatment. Nuclear somatic cell research involves implanting a nucleus from a somatic cell into a human egg cell, (which has had the nucleus removed) so that mitosis produces a blastula, which has the same DNA as the somatic cell donor. The stem cells would then be used for research into how different diseases occur at a genetic level and how they may be prevented or cured2. However, the number of donor eggs required is large and concerns are held over the source of these eggs, for example paying women to donate (Roberts and Throsby 2008), and whether it would lead to human cloning. Recent debates have been held over whether it is right to implant human DNA into animal eggs for similar research which some argue is morally wrong.
The aim of this assignment was to gain a better understanding of the developments of the embryo at a tissue and cellular level. Each stage of the formation of the embryo and beyond is so carefully co-ordinated and even one gene mutation can cause a different protein to be expressed and have an effect on the organism. Gastrulation and neurulation are the main events that take place after fertilization and lay the basic (although still complex) framework for organogenesis of the foetus where all cells need to differentiate and proliferate.