Embryology: 3rd week of development
This article describes the third week of embryonic development.
Every step in the gestational period is crucially orchestrated to ensure appropriate development of the embryo. By the beginning of the 3rd gestational week, the cluster of cells would have differentiated into a bilaminar disc with an amniotic and an exocoelomic cavity. The embryonic disc and cavities are enclosed by the chorionic membrane within the chorionic cavity. If everything went according to plan, then the conceptus would have successfully embedded in the endometrium.
The third week is concerned with establishing left and right handedness, the craniocaudal axis, as well as the conversion of the bilaminar disc into a trilaminar embryo. Additionally, there is further specialization of the extraembryonic structures that will continue to support the embryo during the intrauterine phase. Furthermore, it also happens to be the time of the missed period; which is roughly five weeks following the first day of the previous normal menstruation. This article will focus on the key features of the 3rd gestational week.
- Notochord formation
- Neural tube development
- Lateralization and body axis formation
- Somite formation
- Intraembryonic coelom
- Primitive cardiovascular system
- Chorionic villi
- Gross morphological changes of the embryonic disc
Like many other phases of embryological development, gastrulation is a complex, biochemically dependent process by which the bilaminar embryo acquires a third layer to become a trilaminar disc. It is not uncommon to hear some individuals refer to the embryo as a gastrula during this developmental phase. During this process, the embryo also develops axial inclination. This process also contributes significantly to the morphological changes that the embryo will go through in order to acquire a human shape.
Steps and processes
Gastrulation begins when a linear region of cells of the epiblast layer become thicker at the caudal aspect of the embryo. This primitive (Spemann’s) streak develops as epiblast cells replicate and migrate to the midline of the bilaminar disc under the influence of nodal. Nodal is a transformation growth factor β (TGF β) protein that not only initiates, but also maintains the primitive streak. The streak is comprised of totipotent stem cells from the epiblast that grow in a caudocranial manner. As cells are added to the caudal end of the primitive streak, the cranial end begins to enlarge and forms a primitive (Hensen’s) node.
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.
The cells of the primitive streak synthesize and secrete fibroblast growth factor 8 (FGF8). FGF8 downregulates the expression of E-cadherin, which is intended to promote cellular adhesion. As a result, epiblast cells that have lost their adhesion molecules will subsequently undergo invagination. Not only does this give rise to the previously described depressions (primitive groove and pit), but it also results in migration of epiblast cells between the epiblast and the hypoblast layers. These cells lose their tall columnar appearance and become loosely arranged spindle-shaped cells suspended in collagenous reticular fibers known as mesenchyme. The mesenchyme is made up of pluripotent cells that will provide structural support for the embryo. They also have the ability to differentiate into osteoblasts, chondroblasts, and fibroblasts, in addition to participating in vasculogenesis and angiogenesis.
FGF8 stimulates the expression of another protein, called Bachyury T, which regulates the transformation of other mesenchyme cells to the middle embryonic layer known as the mesoderm. As other epiblast and primitive streak cells migrate deeper, they eventually displace cells of the hypoblast to form the embryonic endoderm. The cells remaining in the epiblast are subsequently referred to as the ectoderm. Therefore, all three germ layers of the gastrula are epiblast derivatives. The mesoderm will eventually separate the ectoderm from the endoderm, except at the points where the two layers are fused (i.e. caudally at the cloacal membrane, and cranially at the prechordal plate). The cloacal membrane is a circular structure that marks the future location of the anus. On the other hand, the prechordal plate gives rise to the oropharyngeal membrane (also a bilaminar region), which will form the future mouth and pharynx.
The three germ layers are responsible for forming all tissues within the body. The fate of each layer is as follows:
- The embryonic ectoderm, which is located on the dorsal surface of the embryo, is subdivided into neural ectoderm and surface ectoderm. The neural ectoderm will give rise to the sensory organs (eyes, internal ears). It also subdivides into the neural tube (which will form the brain and spinal cord) and the neural crest (which differentiates into the head mesenchyme and the peripheral nervous system). The surface ectoderm develops into the epidermis.
- Embryonic mesoderm is a bit more diverse and gives rise to paraxial, intermediate, and lateral plate mesoderm. The lateral plate mesoderm has cardiac, haematological, vascular, and smooth muscle fates. It also gives rise to the spleen, lymphatics, and adipose tissue. Intermediate mesoderm is responsible for the formation of the lower urinary tract, kidneys, and the reproductive system. The paraxial mesoderm first form somites. The somites then differentiate into the rigid structural components of the body (i.e. bone, ligaments and tendons, cartilage and skeletal muscle). They also give rise to the dermis.
- The embryonic endoderm gives rise to the aero-digestive epithelium, as well as the glandular cells of the gastrointestinal tract and its associated organs. The lungs, thymus, thyroid, and prostate glands are also derived from endoderm.
Primitive streak cells continue to migrate and differentiate into mesoderm up until the beginning of the 4th gestational week. As mesoderm production declines, the primitive streak also begins to regress, becoming a small structure in the sacrococcygeal part of the embryo. Eventually, it completely disappears by the end of week 4.
Steps and processes
As the primitive node and streak are formed, invaginating mesodermal cells migrate cranially through the structure. They become prenotochordal cells that travel cranially towards the prechordal plate, in the midline. The primitive pit then projects into the notochordal process, giving rise to the notochordal canal. The notochordal process is now a tubular structure occupying the space between the primitive node to the prechordal plate. The prenotochordal cells of the notochordal process interdigitate with the cells of the hypoblast layer, prior to the invasion of the endoderm. The fused layer subsequently becomes perforated, allowing communication between the notochordal canal and the umbilical vesicle. As these perforations coalesce, the floor of the notochordal canal is lost. This is followed by flattening of the remaining notochordal process to form the notochordal plate.
Proliferation of the notochordal cells at the cranial end of the notochordal plate results in infolding of the tubular structure. Subsequently, a solid cord of cells that is definitively the notochord arises. At the area where the primitive pit descends into the epiblast, the notochordal canal persists; giving rise to a neurenteric canal that provides temporary communication between the amniotic cavity above and the umbilical vesicle below. This communication is obliterated as the notochord detaches from the endoderm.
The notochord projects from the primitive node to the oropharyngeal membrane. As the primitive streak extends caudally, the notochord also follows. Therefore, a craniocaudal growth pattern of the notochord is observed. Also, note that at this stage of development, prechordal mesoderm arises from neural crest cells, just rostral to the notochord. Therefore, the cloacal and oropharyngeal membranes are the only bilaminar regions of the embryo that remain. Notochordal migration is also associated with movement of pluripotent mesoderm that also move cranially, bilaterally with respect to the notochord process and prechordal plate. Once they have established a cranial position in the cardiogenic area of the embryo, the heart primordium is formed from the cardiogenic mesoderm by the end of week 3.
Functions of the notochord
The functions of the notochord in the development of the embryo are as follows:
- It defines the primitive longitudinal axis of the embryo.
- It provides some rigidity to the fragile developing human.
- It contributes to the formation of the intervertebral discs. Fragments of the notochord persist into adulthood as the nucleus pulposus of the intervertebral disc.
- It plays an important role in the formation of the axial musculoskeletal system.
- It is important in the development of the central nervous system.
Neural tube development
Steps and processes
Not only does the notochord influence the epiblast to migrate and form the two deeper layers, but it also stimulates regions of the ectoderm to form the neural plate. This thick, elongated area of epithelial cells is a midline structure is adjacent to the midline and is superficially related – and equal in length – to the notochord. Growth of the notochord corresponds with an increase in width of the neural plate. The plate also grows cranially and meets the oropharyngeal membrane until it eventually outgrows the notochord. There is a prominent invagination along the long axis of the neural plate called the neural groove.
There are raised neural folds on either side of the groove that appear more pronounced at the cranial pole; indicating early brain development has commenced. Inward migration and subsequent fusion of the neural folds occur as the third week comes to a close. The neural plate has now become a neural tube, which serves as the primitive spinal cord and brain vesicles. Some neural crest cells that were not incorporated in the neural tube transform from epithelium to mesenchyme, after which they move away from the fusing neural folds. The underlying neural tube separates from the ectoderm and the non-neural edges of the ectoderm fuse to close the dorsum of the embryo; forming the epidermis.
During the closure of the neural tube, there is dissociation of some underlying neuroectodermal cells at the inner border of the folds. The neural crest cells completely separate from both the surface ectoderm and neural tube during this migration process. They aggregate as flat, irregular cells known as the neural crest that lies between the neural tube and surface ectoderm. The neural crest divides into left and right halves and migrates laterally, to the dorsal region of the embryo (relative to the neural tube).
They then develop into sensory ganglia of cranial (CN V, VII, IX, and X) and spinal (dorsal root) nerves. Other structures that are derived from the neural crest include, but are not limited to:
- Neurilemma sheaths of peripheral nerves
- Leptomeninges of the arachnoid mater and pia mater
- Pigment cells
- The medulla of the suprarenal glands
Importantly, the neural tube contributes significantly to lateralization. The floor of the neural tube expresses the LEFTY protein on the left side of the embryo. Studies suggest that it restricts crossing over of left-sided signals to the right.
Lateralization and body axis formation
While the human body has some amount of symmetry to it (with several organs being paired; each occurring on one side of the body), there are other organs that possess a certain degree of asymmetry and only exist on one side. Lateralization refers to the establishment of left and right sidedness of the body. It occurs as a result of particular genes only being expressed on the left side, and others being silenced on the right.
These events begin prior to the gastrulation phase. The activity of anterior visceral endoderm (AVE) is an example of axial development that precedes gastrulation. The AVE is located at the cranial aspect of the embryonic disc and is integral in the formation of the head. It regulates the expression of LIM homeobox 1 (LHX1), homeobox expressed in ES cells 1 (HESX1), and orthodenticle homeobox 2 (OTX2); which, along with other factors establish the cranial region of the disc. The protein nodal (mentioned previously) also stimulates the production of other gene products that help to define the dorsoventral mesoderm, as well as other cranial and caudal structures.
Bone morphogenetic protein 4 (BMP4) also works in conjunction with fibroblast growth factors to shift mesodermal cells to the ventral region of the body cavity. This ensures that structures such as the body wall mesoderm, blood mesoderm (arising from lateral plate mesoderm) and the kidneys (originating from intermediate mesoderm) will be formed in the ventral area. The impact of BMP4 on the mesoderm is regulated by other genes (noggin, follistatin and Goosecoid) expressed in the primitive node; otherwise, all mesodermal structures would be ventralized.
The Bachyury T gene also participates in the formation of dorsal mesoderm in both the midline and tail region of the embryo. Its gene products are transcription factors that bind to DNA binding domains (T-box), which aid in dorsal and caudal mesoderm formation.
Earlier it was mentioned that nodal is important for the formation of the primitive node and streak. Additionally, these structures also secrete the nodal protein under the influence of FGF8, but only of the embryo’s left side. This is an important factor in the lateralization process. FGF8 induced nodal expression also persists later in embryonal development in the lateral plate mesoderm in addition to the LEFTY-2 (left-right determination factor 2) protein. Both LEFTY-2 and nodal proteins work synergistically to upregulate the expression of another homeobox transcription factor, PITX2 (paired like homeodomain 2) that aids in defining left sidedness. It should be noted that organs found predominantly on the left side of the body (i.e. the primordia of the gut, stomach, heart) are particularly exposed to these left sided proteins.
By the 16th day of gestation, an outpouching extending from the wall of the umbilical vesicle that is adjacent to the connecting stalk develops. This diverticulum is referred to as the allantois. In humans, it is a rudimentary structure that may be linked to pathological processes of the urinary bladder.
The allantoic mesoderm spreads out deep to the chorion and expresses vasculogenic potential; giving rise to the umbilical artery that will supply the placenta (the umbilical vein arises from another source). Proximally, the allantois persists throughout development as the urachus. This structure connects the urinary bladder to the anterior abdominal. In adults, the urachus is known as the median umbilical ligament.
Primitive node tissues are also responsible for the formation of paraxial mesoderm. These are longitudinal blocks of cells that are medially related to the intermediate mesoderm. For completion, the lateral mesoderm is lateral to the intermediate mesoderm, but medial to the extraembryonic mesoderm of the umbilical vesicle and amnion. Under the influence of forkhead transcription factors (FoxC1 and FoxC2), along with NOTCH and HOX genes, condensation and conformational changes of the paraxial mesoderm cells at the end of week three gives rise to paired cube-like bodies of cells called somites.
This development takes place craniocaudally – thanks to the Delta-Notch signalling pathway – on either side of the neural tube. By week four, the embryo will enter the somite period, where around 38 pairs of somites can be observed. This number increases to about 44 pairs by the end of week 5. Somites are unique because they produce marked elevations on the dorsal surface of the embryo. Their prominence also aids in aging the embryo during the fourth and fifth gestational weeks.
A coelom refers to a body cavity. Therefore the intraembryonic coelom is the primitive body cavity within the embryo. Initially, they appear as solitary coelomic spaces in the lateral and cardiogenic mesoderm layers. Subsequent fusion of the spaces forms a solitary, horseshoe-like space that partitions the lateral mesoderm into two layers:
- The splanchnic lateral mesoderm is the visceral layer that is adjacent to the endoderm layer and communicates laterally with the extraembryonic mesoderm of the umbilical vesicle. Together, the two structures will form the splanchnopleure; which is the embryonic gut.
- The somatic lateral mesoderm is the parietal layer that is just deep to the ectoderm and communicates laterally with the extraembryonic mesoderm of the amnion. The two layers combine to form the somatopleure; also known as the embryonic body wall.
The intraembryonic coelom undergoes further division from the 5th gestational week, onwards. At that time, it is divided into the pericardial cavity, peritoneal cavity, and the pleural cavities.
Primitive cardiovascular system
The conversion of a bilaminar embryonic disc to a trilaminar one reduces the efficacy of diffusion as the principal mode of nutrient delivery to the developing cells. Consequently, during the third week, the embryo initiates a more efficient mechanism for nutrient transport and waste disposal. Blood vessels arise by two major mechanisms: vasculogenesis and angiogenesis. The former refers to the formation of new blood vessels via a de novo pathway (i.e. induction and assembly of angioblasts). The latter, however, speaks to the formation of new blood vessels by budding from previously formed vessels. The development of blood itself is referred to as hematogenesis.
Blood and blood vessel formation
Fibroblast growth factor 2 (FGF2) is the primary instigator for vasculogenesis. By binding to fibroblast growth factor receptors (FGFR) on mesoderm, it induces their differentiation into hemangioblasts. These pluripotent cells then aggregate in the yolk sac and give rise to blood islands. With the action of vascular endothelial growth factor (VEGF) acting on vascular endothelial growth factor receptors (VEGF-R2), hemangioblasts eventually differentiate into endothelial cells. VEGF then acts on VEGF-R1 in order to stimulate the characteristic tubular arrangement of the endothelial cells in the blood vessels.
The hemangioblasts that did not contribute to vasculogenesis will have one of two options:
- The peripheral cells can give rise to angioblasts that will participate in angiogenesis.
- Centrally located cells can become hematopoietic stem cells that will differentiate into all blood cells.
Vasculogenesis commences in the extraembryonic mesoderm of the connecting stalk, umbilical vesicle and chorion. It is followed briskly by embryonic vasculogenesis.
Primitive heart formation
As the blood and blood vessels begin to form, there is a concomitant establishment of the cardiogenic area. A pair of longitudinal tubes with endothelium called the endocardial heart tubes are formed during this time. They eventually fuse to form the primordial heart tube. It merges with embryonic and extraembryonic blood vessels to establish the primordial cardiovascular system. In most cases, heartbeats commence at the end of week three, making the heart the first functional organ of the embryo. However, this heartbeat is not readily appreciated until the 5th week of gestation.
The primary chorionic villi first appear as the 2nd week ended. Following this event, they are invaded by mesenchymal tissue as they began to arborize. The now secondary chorionic villi extend across the entire chorionic sac. These mesenchymal cells also have vasculogenic and hematogenic potential and subsequently differentiate into capillaries and blood cells. Once the blood vessels become visible, the structures are called tertiary chorionic villi. There is subsequent fusion of the capillaries that gives rise to the arterio-capillary network. These networks will eventually communicate with the primordial heart tube by the end of week 3.
Fetomaternal exchange of nutrients and waste material can now be facilitated at the villous interface instead of the previous diffusion gradients. The trophoblast also continues to grow such that proliferation of the cytotrophoblast results in projection of the villi through the syncytiotrophoblast. This gives rise to an extravillous cytotrophoblastic shell that encircles the chorionic sac and further embeds into the endometrium.
Gross morphological changes of the embryonic disc
Not only does the embryonic disc now have three layers, but it has also increased in length and (in some areas) width. The previously flat circular disc elongates and becomes broad at the cephalic pole, but slender at the caudal region. The increased width at the cephalic end is attributed to the constant cellular migration to this area.
The invagination migration sequence occurring at the primitive streak continues to progress into the 4th gestational week. It is interesting to note that while there is cessation of gastrulation cranially, the process persists in the caudal region. This phenomenon is related to the fact that cellular specialization in the cranial region precedes that same process caudally. Therefore, the primitive streak continues to undergo growth and promote gastrulation in the caudal segment of the embryo.
The third week of development is certainly not a piece of cake, despite it being a very early phase in the lengthy period of fetal development. The order of the formations that take place during the third week are:
- Germ layers
- Neural tube
- Lateralization & body axis formation
- Intraembryonic coelom
- Primitiva cardiovascular system
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