Riassunto esame Embriologia, Prof. Castagneti Lara, libro consigliato Carlson, Bruce M. Carlson

Letizia Hassan: Fourth week, forming the embryo

Introduction and focus on the heart

During the 4th week, the body plan is established, meaning this week marks the beginning of the organogenesis period, which will last up to the 8th week. In this period, the organ function is minimal except for the cardiovascular system (even if it is still simple, it works properly). During the third week, the embryo attains a size that does not permit simple diffusion to distribute oxygen and nutrients to all its cells or efficiently remove waste products. The early development of the heart and circulatory system is an embryonic adaptation that permits the rapid growth of the embryo by providing an efficient means for the distribution of nutrients. The circulatory system faces the daunting task of having to grow and become continuously remodeled to keep pace with the embryo's overall growth while remaining fully functional in supplying the needs of the embryo's cells.

The heart starts beating around the 21st-22nd day and can be heard through the Doppler ultrasonography from the 5th week.

Key events of the 4th week

The key events of the 4th week are folding (formation of body cavities) and segmentation. This period is also very crucial for the action of teratogens on the embryo. N.B.: A drug used to prevent nausea during pregnancy. The main component is thalidomide, which causes poor growth of upper (if taken during the 4th week) or lower (if taken during the 5th) limbs.

Somitogenesis

The process of somitogenesis starts during the end of the 3rd week but is visible during the 4th. The somites are visible for a short period of time, because later they give rise to several body structures. Only in some parts of the body the effects of segmentation are also visible after birth: the vertebral column (each vertebra originates from a specific somite) and spinal cord.

Macroscopic segmentation takes place only in the trunk region, while the head remains unsegmented, and it follows the order rostral to caudal (guided by the progressive expression of genes, including the HOX genes). The mesoderm of the head becomes more dispersed to loosely fill the developing head as the head mesenchyme. When neural crest cells start to migrate, the head mesenchyme becomes supplemented with neural crest cells (= head mesenchyme derives from both mesoderm and ectoderm).

The formation of individual somites from a homogeneous strip of paraxial mesoderm is a complex process that involves a variety of levels of molecular control and changes in cellular behavior. The first somites form during the 20th day circa at the head-trunk border (always rostral to caudal order). The remainder form at a rate of about three or four a day, finishing on about day 30.

Letizia Hassan: Result 42–44 pairs of somites form but the caudalmost ones eventually disappear, giving a final count of approximately 37 pairs.

The first event to occur is the segmentation of the paraxial mesoderm; it involves two mechanisms incorporated in the clock and wavefront model. The first step (the wavefront) is associated with the elongation of the caudal end of the body through proliferative activity of mesenchymal cells in the most posterior nonsegmented part of the primitive streak. Cells in this area divide actively under the influence of a high local concentration of FGF-8. More anteriorly, where the cells are older, the concentration of FGF-8 decreases as the FGF molecules become broken down over time. Conversely, the cells closer to the last-formed somite become exposed to increasing concentrations of retinoic acid, which is produced in the most posterior somites and whose action opposes that of FGF. At some point, the mesenchymal cells are exposed to a balance of FGF-8 and retinoic acid concentrations that results in the crossing of a developmental threshold (the wavefront, or determination front) that prepares them for entering the process of segmentation (somite formation).

This is characterized by the expression of a transcription factor, Mesp-2, which prefigures a future somite. With the continued caudal elongation of the embryo and the addition of new somites, the location of the wavefront extends caudally in the growing embryo, but it remains a constant distance from the last-formed somite pair.

Next, the segmentation clock is initiated in those presomitic cells that have passed over the previously mentioned threshold and are expressing Mesp-2. The exact mechanism that starts the clock is still not fully defined, but many molecules in the interacting Notch, Wnt, and FGF pathways are known to be synthesized at regular periodic intervals and become localized at critical locations in the forming somite. After, the lunatic fringe becomes concentrated at the future anterior border of the somite, and c-hairy (a homologue of a segmentation gene in Drosophila) becomes concentrated along the future posterior border.

In the picture:

• The wavefront, consisting of opposing gradients of retinoic acid (RA) and fibroblast growth factor-8 (FGF).
• The segmentation clock, in which oscillating molecules in the Notch pathway stimulate the expression of lunatic fringe at the anterior and c-hairy at the posterior border of a future somite. Later interactions between Eph A and ephrin B maintain the intersomitic space.

Letizia Hassan: At the level of cellular behavior, cells at the anterior border of the forming somite express the ephrin receptor Eph A. Because the cells on the posterior border of the previously formed somite express the ephrin ligand ephrin B, the cells of the two adjacent somites are prevented from mixing, and a fissure forms between them.

Finally, the action of wnt-6 from the overlying ectoderm stimulates the expression of the transcription factor paraxis in the newly forming somite. This, along with downregulation of Snail, results in the transformation of the mesenchymal cells into an epithelial cell type (mesenchymal to epithelial transition). In the earliest stages of its formation, a somite also undergoes an internal subdivision into anterior and posterior halves. Eventually, there is a complete transformation of the segmented blocks of mesenchymal cells into a sphere of epithelial cells through the continued action of paraxis.

Somite arrangement

The apical surfaces of the cells surround a small central lumen, the somitocoel (it contains a few core cells), and their outer basal surfaces are surrounded by a basal lamina (containing laminin, fibronectin, and other components of ECM). After, the cells of its ventromedial wall are subjected to an inductive stimulus through signaling molecules shh and noggin (originating from the notochord and the ventral wall of the neural tube); the response is the expression of Pax1 and Pax9 in the ventral half of the somite (= sclerotome).

In this region: burst of mitosis, loss of intercellular adhesion molecules (N-cadherin), dissolution of the basal lamina and transformation of epithelial cells back to a mesenchymal morphology (= EMT creates secondary mesenchyme). These secondary mesenchymal cells migrate or are otherwise displaced medially from the remainder of the somite and begin to produce chondroitin sulfate proteoglycans and other molecules characteristics of cartilage matrix as they aggregate around the notochord.

Under the influence of secreted products of wnt genes produced by the dorsal neural tube and the surface ectoderm, the dorsal half of the epithelial somite becomes transformed into the dermomyotome and expresses its own characteristic genes (Pax3, Pax7, paraxis).

Letizia Hassan: Mesenchymal cells arising from the dorsomedial and ventrolateral borders of the dermomyotome form a separate layer, the myotome, beneath the remaining somatic epithelium, which is now called dermatome (it will contribute to the dermis).

Organization of the somite

By the time three somites have formed behind one, its cells have received sufficient environmental input that their development course is set in place (the fate of the cells is fixed). Sclerotome, for example, can be subdivided into many compartments; eventually, cells from different somatic compartments (ventral, central, and dorsal), come together to form a vertebra. Whereas cells from the central and lateral compartments form the ribs. Later, cells from its medial edge (meningotome) surround the developing spinal cord to form the meninges and their vasculature. Cells of the somitocoel (arthrotome) join with some ventral cells to form the intervertebral disks and the vertebral joint surfaces.

After the Wnt-mediated formation of the dermomyotome, cells in its dorsomedial sector find themselves exposed to a balance of shh signaling from the notochord and Wnt signaling from the dorsal neural tube and overlying surface ectoderm, committing to becoming the myogenic lineage. The myotome will then split again into a dorsal epimere and a ventral hypomere. The epimere forms the deep epaxial muscles of the back (innervated by the dorsal ramus of the spinal nerve); the hypomere forms the hypaxial muscles (lateral and ventral body wall of thorax and abdomen).

Further segmentation details

The spinal cord is divided into segments, and each of these gives rise to two spinal nerves (one on the right and one on the left). The spinal nerves innervate the cutaneous territory (sclerotome), muscular territory (myotome), and the bony-tendinous territory (sclerotome) from the adjacent somite. The neural connection is maintained for life.

Letizia Hassan: Segmentation in the neural tube. Soon after the neural tube has taken shape, the brain region can be distinguished from the spinal cord. The brain region undergoes a series of subdivisions to become organized like an adult brain.

• Early set of subdivisions: 3-part brain (prosencephalon), midbrain (mesencephalon), and hindbrain (rombencephalon).
• Subdivided into: telencephalon, diencephalon, metencephalon, and myelencephalon.

Another subdivision happens through a more subtle level of segmentation (= dividing the brain into transiently visible series of regular segments called neuromeres. The neurosomes in the hindbrain are also called rhombomeres (visible from early 4th week to late 5th week). The midbrain doesn't seem to be segmented, but the prosencephalon contains fewer regular series of prosomeres. Rhombomeres are arranged as odd and even pairs and act as isolated compartments, providing the basis for the fundamental organization of the hindbrain. Cells of adjacent rhombomeres do not intermingle (between odd and even), but cells of side by side odd-odd or even-even rhombomeres do intermingle.

How segmentation occurs

The neural tube is subjected to vertical inductions from the notochord and head organizing regions (anterior visceral endoderm and prechordal plate), important in inducing the forebrain region. Another mechanism is the gradient of Wnt-8 signaling, which effectively subdivides the regions of the brain (forebrain/midbrain and hindbrain/spinal cord segments). This happens thanks to the help of two transcription factors, Oxt-2 in the forebrain/midbrain region and Gbx-2 in the hindbrain. The midbrain-hindbrain border becomes an important signaling center, the isthmic organizer. Wnt-1 is synthesized in the neural ectoderm anterior, and FGF-8 is formed posterior to the isthmic organizer. Fundamental in organizing the development of the midbrain and the cerebellum are the transcription factors (expressed on both sides of the isthmic organizer) Pax-2, Pax-5, and engrailed. There are two other important signaling centers: the anterior neural ridge (anterior pole of the brain).