Third Week of Development (Trilaminar Germ Disc)

The third week of embryonic development is featured by cells of the bilaminar embryonic disk – made of an epiblast and a hypoblast- undergoing a highly complicated process of differentiation and rearrangement, called gastrulation. During this process, the two cell layers  of the bilaminar embryonic disc transform into three layers known as the three germ cell layers. In addition, the cephalocaudal body axis is established.

Gastrulation occurs in the third week of human embryonic development; it is a critical process that generates the three primary germ layers, the ectoderm, mesoderm and endoderm. Gastrulation transforms the embryo from a one-dimensional layer of cells into a multidimensional multilayered embryo called the gastrula. It sets up the embryo for organogenesis and provides mechanisms for forming a multileveled body plan and anatomical axes formation. These axes are the craniocaudal axis also known as the superior-inferior  axis, and rostro-posterior which is also called the antero-posterior axis.

We have to remember that embryonic development is a continuous process over time. As seen in the previous week (second week of development), cells of the epiblast yield the amniotic sac, whereas a few cells of the hypoblast extend downwards and proliferate to surround the cavity of the primitive yolk sac forming the definitive yolk sac. Then, a raised area of columnar cells develops on the hypoblast. This raised area is called the prechordal plate; it lays the earliest step in defining the cranial end of the developing embryo. Thereafter, the primitive streak, which is an important structural feature and a landmark in the developing embryo at this stage, appears. The primitive streak is the fulcrum of the process gastrulation; it plays a significant role in the generation of the three germ layers.  Cells of the epiblast migrate towards the primitive streak, undergo an epithelial to mesenchymal transition and dip into the primitive streak on their way to form other new layers, the mesoderm and the endoderm. Accordingly, gastrulation is a process of cellular rearrangement which involves migration of epiblast cells, their invagination at the primitive streak and subsequent differentiation.

The primitive streak is a longitudinal present in caudal part of the midsagittal line of the embryonic disc. It firmly establishes the craniocaudal axis of the body. As cells of the epiblast proliferate and migrate toward the midline of the embryo, they form a thickening that elongates to become linear in shape, thus called the primitive streak.

The cranial end of the embryo seems to play an important role in initiating the process of gastrulation and leading the way in embryonic development. Epiblast cells present in the cranial end of the primitive streak ingress (plunge) into the streak at a greater speed forming a circular cavity known as the primitive pit. Meanwhile, the primitive streak elongates and migrating epiblast cells join the streak at the cranial end, forming a mass of cells around the primitive pit. This mass is called the primitive node or Hensen’s node, which at this stage is the primary tissue organizer, where transcription factors and chemical signals induce tissue formation. Several signaling factors are involved in the complex differentiation of the primitive streak; these factors include the transforming growth factor-beta (TGFB) and the bone morphogenetic protein (BMP), Vg1 protein, Nodal, Wnt-8 protein, fibroblast growth factor 8 (FGF8) and Chordin. The plunging of the epiblast cells into the primitive streak is called ingression. The first epiblastic cells to migrate ingress across the primitive streak integrate with the hypoblast and arrange themselves to form a new layer known as the endoderm. Ingression of epiblastic cells across the primitive streaks continues and a new third layer is formed in the middle; this middle layer is known as the mesoderm.

Fig. 6.1. The illustration show feature of the primitive streak as seen in a whole mount (left) and in a 3-D transverse section of the embryonic disc. Cells epiblast (future ectoderm) migrate medially towards the midline and ingress (curved to arrows) forming the primitive streak, the endoderm and the mesoderm. The red arrows shows directions of cell migration. The wholemount illustration on the left shows Hensen’s node as a small spherical collection of cells.

Epiblast cell migration towards the primitive streak continues from all directions to ingress down the primitive steak to a level below the epiblast and then migrate and spread in the opposite direction.  In this way the endoderm and the mesoderm are formed as cell sheets underlying the epiblast. The endoderm is the first of the three germ layers to be established; the mesoderm is the second germ layer to establishes, it occupies the space between the epiblast and the endoderm.

The primitive streak is made of a groove known as the primitive groove flanked on each side by a ridge known as the primitive ridge. Cells moving medially during gastrulation in the epiblast compile to from the primitive then dip into the primitive groove. At the base of the primitive groove they reverse direction to the endoderm and mesoderm.

Fig. 6.2. The illustration shows the primitive groove, the primitives ridges and directions of cell migrations (blue arrows) in  the epiblast and in the newly formed endoderm and mesoderm. Ingression of cells of the epiblast into the primitive groove is shown by red arrows.

The primitive node, which is also called Hensen’s node, is an accumulation of organized cells present in the cranial end of the primitive streak. It forms the cranial boundary of a small circular hole known as the primitive pit. This hole is formed as a result of the higher speed by which the migrating cells of the epiblast cells ingress at this point as compared to the ingression speed in other parts of the primitive streak. The primitive streak yields the embryonic endoderm, the notochord, the embryonic mesoderm and the extraembryonic mesoderm.

Fig. 6.3. The illustration shows the origin and main derivatives of the primitive streak.

The primitive streak is made of totipotent stem cells that can proliferate and differentiate to give rise to all types of embryonic and extraembryonic tissues.

The primitive node (and streak) is maintained by the hepatocyte nuclear factor 3β (HNF-3 β; a product of the FOXA2 gene). The presence of this protein is also crucial for the formation of forebrain and midbrain structures as well. Simultaneously, a slender depression develops within the streak that is continuous with the sunken area at the primitive node (i.e. the primitive groove and primitive pit, respectively). The establishment of these structures allow identification of the cranial (near the primitive node) and caudal (towards the tail of the primitive streak) poles of the embryo. It also facilitates the identification of the left and right sides, as well as dorsal and ventral surfaces of the embryo.

Major Derivatives of the Primitive Streak

The primitive streak plays a pivotal role in embryonic development in humans and other vertebrates. It is the main organizer of formation of the germ layers. Its derivatives are diverse including the various tissues and organs of the body. To begin with, it gives rise to the endoderm and mesoderm.

Derivatives of the endoderm  include the epithelium of the alimentary tract, the respiratory tract, the thyroid gland, the liver and the pancreas.  It also gives rise to gastric and intestinal glands.

The mesoderm differentiates into four parts, the axial, paraxial, intermediate plate and lateral plate. The axial mesoderm arises in the midline of embryonic disc cranial to the primitive pit. It has two parts, the notochord which contributes to the formation of the vertebral column, and the prechordal plate which contributes to the formation of the head.

The paraxial mesoderm flanks the axial mesoderm (the notochord) on either side. It later on differentiates into masses known as somites; somites are a characteristic feature of the developing embryo when the primary germ layers are forming during later stages of gastrulation. The somites are arranged segmentally in pairs and differentiate into myotome which develops into skeletal muscle, dermatome which develops into the skin and sclerotome gives rise to the vertebral column and ribs.

The intermediate mesoderm arises from main organs of the urogenital system including the kidneys, testis, the ovaries and adrenal glands.

The lateral plate mesoderm is unique in that it splits into two layer known as the somatic mesoderm and the splanchnic mesoderm. The heart, blood vessels, and blood cells develop from the lateral mesoderm.

Fig. 6.4. The illustration shows the location of mesoderm, and its four parts, namely the axial mesoderm (notochord), the paraxial mesoderm, the intermediate mesoderm and the lateral mesoderm. The mesoderm is sandwiched between the ectoderm (above) and the endoderm (below).

Molecular Aspects of Primitive Streak Development

The primitive streak is a transient inductive tissue that is affected by several key signaling molecules, including Wnt3, BMP4, and Nodal. It is the point where cells of the epiblast under the influence of these factors converge in a well-defined spatial and temporal sequence. The primitive streak is the organizing center for gastrulation, which defines the future embryonic midline and serves as a conduit of cell migration for germ layer formation. The key factors involved induction and regulation the primitive streak formation include Vg1, Nodal, Wnt8C, FGF8, TGFB, E-cadherin and Chordin.

Vg1 is a gene that encodes growth factor protein (VG1) related to the transforming growth factor beta (TGF- β) family. It was discovered in the vegetal cytoplasm of oocytes. It is a cell-signaling factors and is  associated with formation of the mesoderm, the dorsal-ventral axis, and in left-right development. TGF- β belongs to a group of multifunctional cytokines of transforming growth factors and other signaling proteins.

Wnt factor is a factor that regulates cell growth, cell motility, and differentiation during embryonic development. Wnt acts in a paracrine fashion by activating signaling cascades inside the target cells. It is an important factor for cell renewal.

Nodal, a morphogen that belongs to the transforming growth factor ß (TGF-ß) family; it is essential for stabilization of the epiblast state. Nodal protein factor a paracrine effect with an extremely short range limited to the immediate neighborhood of the source cell. Nodal factor is essential for induction of both the anterior-posterior axis, left-right axis, and specification of the mesoderm and endoderm.

Fibroblast growth factors are abbreviated as FGF and are of many subtypes. FGF-4 is an important growth regulator for stem cells, fibroblasts, and endothelial cells. FGF-8  factor (protein) is involved in many processes, including cell division, regulation of cell growth and maturation, and embryonic development.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGF- β) superfamily. BMP factor present in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites. Chordin is a protein factor that plays a prominent role in dorsoventral patterning during early embryonic development. It is essentially a dorsalizing protein factor that binds with a vernalizing protein known as TGF-β. Chordin is a BMP inhibitor that blocks BMP from binding with its receptors in responsive cells.

Fig. 6.5. The illustration shows the signaling factors Vg1, Wnt, Nodal, Chordin, BMP, and FGF that are involved in the epiblast differentiation and development of the primitive streak.

Cells of the primitive streak synthesize and secrete fibroblast growth factor 8 (FGF-8). FGF-8 downregulates the expression of E-cadherin, which promotes cellular adhesion. As a result, epiblast cells lose their adhesion molecules and undergo invagination. This results in the formation of the primitive groove and the primitive pit and also results in migration of epiblast cells between the epiblast and the hypoblast layers forming the mesoderm. During this process, the epiblast cells lose their tall columnar appearance and become loosely arranged spindle-shaped cells known as mesenchymal cells. The mesenchymal cells are pluripotent cells that provide structural support to the developing embryo. They differentiate into a wide range of supportive cells. FGF8 stimulates the expression of another protein, called Brachyury-T, which regulates the transformation of mesenchyme cells into cells of the mesoderm.

The primitive streak is divided into different regions, each with distinct gene expression profiles. Gene expression studies have identified several key genes involved in the development of the primitive streak including Brachyury (BRA), Chordin (CHD) and WNT-8. BRA marks the general mesoderm precursor territory, whereas CHD defines the dorsal mesoderm precursors.  WNT-8 defines the ventral mesoderm precursors.

As epiblast and primitive streak cells migrate deeper, they displace cells of the hypoblast to form the embryonic endoderm. Those cells that remain in the epiblast after formation of the mesoderm and endoderm constitute the ectoderm. Hence, the three germ layers formed during gastrulation are derivatives of the epiblast. The mesoderm separates the ectoderm from the endoderm, except at the points where the two layers are fused caudally and cranially at the prechordal plate.

Regression of the Primitive Streak

The primitive is formed during gastrulation in the caudal part of developing embryo. It continues to elongate and expand cranially until the 18thday postfertilization. Thereafter it regresses and disappears marking the end of the process of gastrulation and the beginning of organogenesis. Regression of the primitive streak begins after formation of the intra-embryonic mesoderm, and the streak completely disappears by the end of the fourth week of embryonic development. By the 22^nd day, the primitive streak regresses to about 20% of its initial length and by the end of the 26th day it disappears altogether. Regression of the primitive streak takes place in an orderly manner. When regression of the primitive streak is complete, its remnant constitutes a poorly organized mass of cells located at the posterior end of the embryo known as the tail bud or caudal eminence that later gives rise to posterior regions in the body. At the same time the notochord develops cranially from the primitive node (Hensen’s node). The notochordal process grows longer until it fuses with the endoderm to form the notochordal plate. Once the fusion is complete, there is a free passageway between the amniotic cavity and the yolk sac, known as the neuromeric canal. This canal maintains a pressure equilibrium between the amnion and the yolk sac.

Several molecular mechanisms control the regression of the primitive streak. Wnt signaling pathway plays a critical role in regulating the regression of the primitive streak. Likewise, BMP signaling pathway is also involved in the regression of the primitive streak's. Moreover, FGF signaling pathway also participates in the process of regression of the primitive streak.

Abnormal regression of the primitive streak can lead to developmental anomalies and developmental delays including delays in the formation of organs (delayed organogenesis).

Fig. 6.6. The illustrations demonstrate the process of regression of the primitive streak. Illustrations of the left shows the primitive streak fully developed with the primitive node and the primitive pit at its cranial end. The illustration in the middle shows the notochord develop cranially from the primitive node. This is accompanied by the start of regression of the primitive streak. The illustration on the right shows that the notochord is well developed flanked by somites and the lateral mesoderm. At this point the primitive streak has greatly regressed with the primitive eminence appearing at its caudal end.

Cell Differentiation

Cellular differentiation is a key process in the development of the embryo and thereafter. Embryonic development is brought about by cellular proliferation and differentiation. Cell differentiation is the process by which a cell changes structure and functions to become more specialized to perform a specific role in the body. It is the transition of a cell from one type into another cell type. It involves a switch from one pattern of gene expression to another pattern of gene expression. Thus, differentiation involves both genotypic and phenotypic cellular changes. Cell differentiation is an intricate process whereby an unspecialized cell transforms into a more specialized cell.  Cell differentiation is crucial for embryonic development and maintenance of tissues and organs.

Cell differentiation is brought about by changes in gene expression induced by signaling molecules in the environment that activate or repress different transcription factors that are necessary to express certain genes in the DNA. The process of cell differentiation involves the following.

Determination is where the cell becomes committed to a specific developmental pathway. Determination of a cell to a particular fate can be broken down into two states, a state where the cell is specified (committed) and state where the cell is determined. In the state of being specified, the cell type is not yet determined and any preference the cell has toward a certain fate can be stopped and reversed or transformed into another fate.

Differentiation is where the cell actually undergoes changes in gene expression that lead to the obtaining specialized structural features and functions. During this stage changes in gene expression, these changes are mediated by transcription factors and epigenetic modifications. In this process, signaling pathways such as Notch and Wnt play critical roles in regulating the process of differentiation. Furthermore, cellular interactions with neighboring cells and the extracellular matrix modulate the process of differentiation.

Maturation is the stage of differentiation where the cell reaches its final functional state.

Thus, it is apparent then that cellular differentiation is a complex process that enables cells to acquire specialized structures and functions, ultimately giving rise to the diverse range of cell types in the body.

Induction in Embryology

Induction is an important process in embryonic development, where one cell or a group of cells influences the developmental fate of another group of cells; the first type of cells is known as the influencer cells and other type is known as the responder cells. this means that certain types of embryonic cells have the potential to direct the differentiation of adjacent cells. This process allows for the formation of complex tissues and organs.

The mechanism of Induction involves signaling pathways, transcription factors and cell to cell interactions. Signaling pathways that need to be activated include Wnt, BMP, and FGF. Transcription factors that play significant roles in induction include Sox and T-box (Tbx), that play critical roles in regulating gene expression during induction. Cell to cell interactions occur between adjacent cells and are known as direct cell to cell interactions; they are mediated by biomolecules such as cadherins and integrins.

A good example of induction is the one that takes place during development of the eye. The optic vesicle which arises from the forebrain approaches the overlying head ectoderm and induce the ectodermal cell with which they come in contact to differentiate in a specific pathway to develop into the eye lens.

Fig. 6.7. The illustration shows the process of embryologic induction. The optic vesicle approaches the overlying head ectoderm and induces its cells to differentiate into the lens placode that develops into a lens pit, a lens vesicle and ultimately form the eye lens.

 Cell Potency

In general potency is the ability or capacity to achieve or bring about a particular result. Cell potency is the ability of a cell to differentiate into other cell types. Cells are classified into different types depending on this ability. Some embryonic cells are capable of differentiating into all sorts of body cells, these are known as totipotent cells; they include the blastomeres of morula and the inner cell mass of the blastocysts. Some other cells are capable of differentiating in a very wide array of cell types but not all cells of the body. These are known as pluripotent cells; they include cells of the ectoderm and mesoderm. Some other cells are able to differentiate into several types of cells and are known as multipotent cells; these include neural stem cells and intestinal stem cells. Finally, there are some other stem cells that give rise to only one type of functional cells; these include germ stem cells, oogonia and spermatogonia which differentiate into sperms and ova. Epidermal stem cells in the skin are also unipotent cells; they differentiate into one type of cells known as keratinocytes.

Fig. 6.8. The illustration shows stem cell differentiation capabilities.

Embryonic Stem Cell Therapy

Stem cell therapy, which is also called regenerative medicine or cell replacement therapy, is a branch of science that aims at promoting the repair of injured or diseased tissues using stem cells. It is reminiscent of organ transplantation but uses cells instead of organs. Stem cells are either embryonic stem cells or adult stem cells; they are both utilized for regenerative medicine. Stem cells as mentioned above have different proliferation and differentiation capabilities; some are pluripotent, some are multipotent, and others are unipotent. All different types are utilized for cell replacement therapy. Stem cell therapies provide patients with promising therapeutic benefits in different disease areas.

Adult stem cell therapy is not as versatile and durable as embryonic stem cells. Often, adult stem cells cannot be easily manipulated to produce different types of functional cells; this to an extent limits utilization of adult stem cells for cell replacement therapies.

Embryonic stem cell, particularly those obtained from morulae and blastocysts have profound capabilities for cell replacement therapies being totipotent or pluripotent. The most significant potential limitation of embryonic stem cell therapy is the problem of immune rejection. Other important limitation are the ethical issue, carcinogenicity, genetic instability and technical difficulties.

Embryonic stem cells are often harvested from blastocysts 5-days old containing about 150 pluripotent stem cells and implanted or injected into the patient’s affected organ e.g. heart, liver, or pancreas. The injected or implanted embryonic stem cells will proliferate and differentiate into normal functional cells that replace the affected cardiac myocytes, hepatocytes or pancreatic beta cells. As a result, the organ is expected to regain its normal function. Thus, embryonic stem cell therapy has great potential applications including treatment of cardiac myopathies, diabetes, multiple sclerosis, Parkinson’s disease, and spinal injuries. Embryonic stem cells can also be utilized to create personalized cell lines for individualized treatment.

Fig. 6.9. The illustration shows the procedures for harvesting embryonic stem cells.

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