Introduction

This article will give a brief overview of gastrulation, a critical process during week 3 of human development. Gastrulation is defined as an early developmental process in which an embryo transforms from a one-dimensional layer of epithelial cells (blastula) and reorganizes into a multilayered and multidimensional structure called the gastrula. In reptiles, avians, and mammals, which are triploblastic organisms, gastrulation derives a three tissue-layered organism composed of endoderm, mesoderm, and ectoderm; each germ layer corresponds to the development of specific primitive systems during organogenesis.  In addition to setting the embryo up for organ formation, gastrulation provides a mechanism to develop a multileveled body plan that demarcates anatomical axis formation with dorsal/ventral and cranial/caudal axis (also termed anterior or rostral/posterior, respectively), retention of global left/right symmetry, and the loss of bilateral symmetry in specific systems (e.g., heart). 

Development

After fertilization, the single-celled zygote will undergo multiple mitotic cleavages of the blastomeres to change from a two-celled to a 16-celled ball or morula. The morula begins as a solid mass of totipotent blastomeres but then undergoes compaction and cavitation to transform into the blastula (non-mammalian term) or blastocyst (human development).  Within the blastocyst, two tissue layers differentiate: an outer shell, known as the trophoblast, and an inner collection of cells termed the inner cell mass (ICM). Cells within the outer ring/shell bind together via gap junctions and desmosomes to undergo compaction, which ultimately forms a water-tight ring/shell called the trophoblast.[1] The outer trophoblast will develop into structures that provide nutrients, help the growing embryo implant in the uterine lining, and become part of the placenta. Additionally, the trophoblast cells are essential in the cavitation of the solid morula into a hollowed-ball of cells with an internal cavity. Trophoblast cells utilize the active transport of sodium ions and osmosis of water to form a fluid-filled cavity known as a blastocoel.[2] 

The cells remaining after cavitation/blastocoel formation are pluripotent ICM progenitor cells, which will give rise to the distinctive formation of the fetus. Rather than being an arrangement of a solid sphere of cells, the inner cell mass is pushed off to one side of the sphere formed by the trophoblast. Together the trophoblastic layer, blastocoel, and ICM define the human blastocyst.[3] From zygote to blastocyst formation, the organism has been surrounded by the zona pellucida, which is a layer of the extracellular matrix that plays a role in the protection and prevention of implantation into the uterine tubes. During blastocyst formation, the zona pellucida begins to disappear from the blastocyst, allowing the ball of cells to proliferate, differentiate, change shape, and eventually implant into the uterine wall. 

During implantation, the trophoblastic layer, which surrounds the blastocyst, further differentiates into two functionally distinct layers. The outer trophoblast, known as the syncytiotrophoblast, releases digestive enzymes to assist with implantation to the endometrium. This layer also releases human chorionic gonadotropin (hCG, necessary in regulating progesterone secretion), the protein used in many pregnancy tests.[4] The inner trophoblast layer, known as the cytotrophoblast, is a single sheet of cells surrounding the extraembryonic mesoderm. Within the cytotrophoblast is the ball of ICM, and during the second week of human development, the ICM cells spread into a flattened tissue layer and differentiate into a two-layered tissue containing epiblast  (columnar epithelial cells) and the hypoblast (cuboidal epithelial cells), which are together known as the bilaminar disc.[5] The formation of the bilaminar disc sets the dorsal/ventral axis as the epiblast cell layer is positioned dorsal to hypoblast. The anatomical location of the bilaminar disc is found between the amniotic cavity and the primitive yolk sac.  The cells of the epiblast stretch to form a semi-sphere known as the amniotic cavity, while the cells of the hypoblast extend to surround the yolk sack. On the hypoblast is a raised area of columnar cells known as the prechordal plate; this is the earliest delineation of cranial from caudal. Development of the bilaminar disc directly precedes gastrulation, where the end goal during week 3 of development is to transform the human blastocyst into a multilayered gastrula with endoderm, mesoderm, and ectoderm.

Cellular

The beginning of gastrulation is marked by the appearance of a groove in the caudal end of the epiblast layer known as the primitive streak.[6] Thus, the formation of primitive steak firmly establishes the cranial/caudal axis. The primitive streak initially forms via a thickening of cells near the connecting stalk. As cells proliferate and migrate toward the midline of the embryo, the thickening elongates to become linear in shape, thus the term primitive steak. The cranial end of the embryo seems to play an important role in beginning the process of gastrulation. At the cranial end of the primitive streak, epiblast cells ingress at a greater rate forming a circular cavity known as the primitive pit. As the primitive streak and pit elongate, migrating epiblast cells join the streak at the cranial end, forming a mass of cells called the primitive node, which becomes the primary tissue organizer where transcription factors and chemical signaling drive induction of tissue formation. Known factors in primitive streak formation include TGFB, WNT, Nodal, and BMPs and are discussed in more detail in the molecular section. 

Epithelial cells in the lateral edge of the epiblast layer undergo an epithelial to mesenchymal cellular transition to delaminate (detach) and migrate down/into the primitive streak.[7] The movement of epiblastic mesenchymal cells down primitive streak is known as ingression.  The first set of cells to move down primitive streak integrate into the hypoblast layer and transform into endoderm, the first of the three germ layers.  The second set of cells to detach and ingress will fill in the space between the endoderm and epiblast layer to form the second germ layer termed mesoderm.  Multiple mesodermal structures will develop: cells that move into the body stalk will help to form extraembryonic mesoderm, and later the umbilical cord, cells passing through primitive pit become notochord or paraxial mesoderm, and other cells coming through streak become lateral plate or extraembryonic mesoderm.  Finally, the remaining epiblast cells will transform into the final germ layer, ectoderm. Cell proliferation and ingression continue in all directions as the embryo grows; however, the primitive streak will always expand directionally from the caudal to the cranial end and then regress in the opposite fashion. Regression occurs after the formation of the intra-embryonic mesoderm, and the primitive streak should completely disappear by the end of the fourth week — a lack of primitive streak regression results in clinical abnormalities.

After the three germ layers have formed, the newly produced structure (trilaminar disc or gastrula) is primed for organ system formation, which is highly reliant upon direct interaction/communication and induction events between the endoderm, mesoderm, and ectoderm.  Cells continue to invaginate through what is now called the primitive node. The cells begin to form a hollow tube extending from the cranial end to the prechordal plate, known as the notochordal process. As the embryo continues to grow in each direction, 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 neurenteric canal.[8] It is theorized that the neurenteric canal forms as a way to maintain pressure equilibria between both chambers. Later in development, the two edges of the notochordal plate will then fuse, becoming a solid mesoderm rod known as the notochord.  The notochord is one of the most important features in embryology. It is a mesodermal structure that not only provides structural support but marks the midline of the embryo. It will provide chemical and physical interactions with the dorsal lying ectoderm to specialize a portion of that ectoderm into neuroectoderm to derive the nervous system.

Biochemical

RNA helicase A (RHA) can function as a helicase with both RNA and DNA. The sequence and biochemical conservation of RHA and its homologs to humans suggests an evolutionarily conserved function. Normal gastrulation depends on RNA helicase A activity, as lack of proper RHA signaling results in ectodermal cell death with clear alterations in differentiation.[9]

The differentiation of pluripotent stem cells to lineage-specified cells within the endoderm, mesoderm, and ectoderm is marked by down-regulation of pluripotency markers (Oct 4, Nanog, Sox 2, etc.) in conjunction with the activation of lineage-specific gene expression, including microRNAs. MicroRNAs (miRNAs) have demonstrated to be enriched in germ layers, specifically targeting TGFB to promote mesoderm and restrict or block neuroectoderm.[10]

Molecular

Primitive Streak

The initiation of the primitive streak is based upon a system of signaling pathways working to both positively and negatively regulate downstream expression. The combination of TGFB, WNT, Nodal, and BMPs are all important in primitive streak development.[11][12][13][14][15] The interplay between Wnt and TGFB signaling seems to be the inducer of the formation of the primitive streak. Specifically, Vg1 (a member of the TGFB family) has been shown to induce streak formation and to prevent formation with Vg1 misexpression at the posterior marginal zone.[16] Vg1 acts on Nodal to continue the chemical cascade to streak formation. To ensure the proper location of the streak on the epiblast, the hypoblast releases antagonists of Nodal signaling.[17] Additionally, the induction of streak formation can be regulated by Wnt factors; not only has upregulated Wnt induced streak formation, but the use of Wnt antagonists such as Dkk-1 and Crescent prevents the formation of the streak.[11] Finally, BMP signaling has been shown to regulate streak formation. Towards the streak itself, BMP concentration is low, with the surrounding embryo exhibiting higher levels of active BMP.  In addition to this, BMP inhibitors cause the formation of a streak in chick embryos. As seen in BMP and other signaling, concentration gradients are typical through most of the gastrulation process, where the different concentrations of signaling factors allow for cells to differentiate into unique tissues.

Endoderm

Endoderm is the embryonic precursor to the thyroid, lungs, pancreas, liver, and intestines, which evolve from four consecutive steps developmental steps: proliferation and induction of pluripotent stem cells, the separation of stem cell-derived endoderm versus mesoderm germ layers, anterior-posterior patterning, and bifurcation of liver and pancreas.  Cells near the anterior portion of the primitive streak will express Forkhead box A2 (Foxa2) to become definitive endoderm (DE). The DE will pattern itself into the foregut, midgut, and hindgut via mesodermal induction during embryonic folding with foregut cells expressing Hhex, Sox2, and Foxa2 and the hindgut expressing different homeobox genes Cdx1, Cdx2, and Cdx4.  The upregulation of TGF-beta signaling promotes pancreas formation with BMP and FGF/MAPK signaling to specify the liver.[18] The specification of the respiratory bud starts with the expression of the Nbx1-2gene. Complex signaling between the respiratory bud epithelium and mesoderm involves FGF and FGFR interactions to promote the growth of the respiratory bud. [19]

Mesoderm

Epiblast cells invaginating through the primitive streak that express high levels of a fibroblast growth factor (FGF2) are fated down a path towards becoming mesodermal cells, but more specifically, they will end up paraxial, intermediate, or lateral plate mesoderm, which will correlate to different tissues as the embryo continues to develop.[20] 

Notochord

Progenitor cells from the node/pit migrate to initiate notochord formation along with epiblast cells from the floor plate of the amniotic cavity filling in the notochord to form a thick rod-like structure down the midline of the embryo. Providing support and serving as an induction center for surrounding cells, the notochord in vertebrates extends throughout the entire length of what will be the vertebral column and reaches as far as the midbrain. Notochord develops first, and then mesodermal cells grow medially to surround it. The notochord is only present in developing organisms with the primary goal of patterning the tissues surrounding them.  Notochord secretes Sonic Hedgehog, Chordin, and Noggin in a morphogenic gradient pattern (highest concentration is near the notochord with diffusion outward), which binds to receptors on target cells to induce specification and differentiation events in the neural plate, somites, and ectoderm.[21]

Mesoderm divides into three main categories: (par)axial, intermediate, and lateral mesoderm, which are the embryonic precursors to a large variety of cells and tissues, including smooth, cardiac, and skeletal muscle, kidney, reproductive organs, the muscles of the tongue, and the pharyngeal arches muscle, connective tissue, bone, cartilage, dermis and subcutaneous layer of the skin, dura mater, vascular endothelium, blood cells, microglia, and adrenal cortex.

• Paraxial mesoderm-Paraxial mesoderm cells first organize to form somitomeres. As the somitomeres develop into somites in a cranial to caudal fashion, the outer cells undergo a mesenchymal to epithelial transition, which serves as a distinct boundary between individual somites. Individual somites then separate into cranial and caudal portions, followed by the cranial portion of each fusing with the caudal portion of the somite directly anterior to it. Distinct regions of each somite (sclerotome, dermatome, myotome) become specific tissue and cell types as the body matures. The skull, vertebral column, and brain meninges develop from the mesoderm surrounding the neural tube and notochord.
• The intermediate mesoderm connects the paraxial mesoderm with the lateral plate and differentiates into urogenital structures.
• Lateral plate mesoderm splits into parietal (somatic) to aid in lateral body fold wall formation, and visceral (splanchnic) layers are involved in gut tube formation. 

Ectoderm

The interplay between bone morphogenic proteins (BMP’s) and Hox genes is integral to the differentiation of the remaining epiblast tissue into the ectoderm. This is especially important in regards to what will become the neuroectoderm, setting up the brain and spinal cord, as well as the surface ectoderm.[13]

The notochord is the main inductive tissue to delineate neuroectoderm from the remaining ectoderm that will become skin.[22] The entire presumptive ectoderm plate expresses BMP and TGF-beta. Noggin and Chordin secretion from the notochord diffuses into the ectoderm directly anterior to the notochord and binds to receptors in the overlying ectoderm to block BMP.  The blockade of BMP specifies the tissue to neural ectoderm, while the remaining ectoderm, which still expresses BMP, will become skin.

Function

Gastrulation occurs during week 3 of human development. The process of gastrulation generates the three primary germ layers (ectoderm, endoderm, mesoderm), which primes the system for organogenesis and is one of the most critical steps of development. The endoderm is the innermost layer, which gives rise to the gastrointestinal tract, the lining of the gut, liver, pancreas, and portions of the lungs and glandular tissues. The mesoderm derives the musculoskeletal system, including connective tissue, the non-epidermal portions of the integumentary system, the circulatory system, the kidney, and the internal sex organs. The ectoderm is the outer layer of the embryo, which gives rise to the external ectoderm (epidermis, hair, nails) and the neuroectoderm (neural crest and neural tube-brain and spinal cord), along with the lens of the eyes and the inner ear. Another important function of gastrulation is to establish directionality within the developing embryo. Cranial/caudal directionality becomes established by the placement of the prechordal plate and the path of the primitive groove, and the establishment of the dorsal/ventral axis is by the layering of the epiblast and hypoblast (discussed above).

Mechanism

Gastrulation involves a complex series of cellular morphogenesis, cellular movements, and cell signaling via transcription factors, chemical morphogenic gradients, and differential gene expression to allow for the induction of germ cell layer formation that orchestrates the initiation of eventual organ system development.

Clinical Significance

Miscarriage is the most frequent type of pregnancy loss, according to the American College of Obstetricians and Gynecologists. Spontaneous abortion is defined as embryonic or fetal death or passage of the products of conception before 20 weeks gestation, with early miscarriage occurring in the first thirteen weeks. Estimates are that 10% to 25% of all clinically recognized pregnancies will end in miscarriage.[23][24] Investigators report that most defects related to gastrulation are incompatible with life, with select instances where mothers can carry fetuses with associated teratologies to term. Morphogenetic processes occurring between the blastocyst stage and gastrulation can be altered and result in structural abnormalities, including patterns of multiple congenital anomalies (MCAs) arising from developmental field defects. Severe damage may cause the death of the product of conception, or embryonic stem cells may repair the damage and allow development to continue.

Teratoma is defined as a solid mass or germ cell tumor comprised of a combination of tissues from all three germ layers. Teratoma is often a result of abnormal persistence (or lack of full regression) of primitive steak. The most diagnosed fetal teratomas are sacrococcygeal teratoma (Altman types I, II, and III) and cervical (neck) teratoma.[25]  Because these teratomas project from the fetal body into the surrounding amniotic fluid, they can be visible during routine prenatal ultrasound exams. Teratomas within the fetal body are less apparent on ultrasound; for these, MRI of the pregnant uterus is more informative.

Sirenomelia (also known as caudal dysgenesis or mermaid syndrome) is a congenital deformity consisting of a total or partial fusion of the legs, often accompanied by urogenital and gastrointestinal malformation.[26] Sirenomelia incidence rates in the literature range from 1 per 60,000 to 1 per 100,000 and have a male to female bias.[27]  There are two theories regarding the development of this pathology. The first is the vascular steal theory, in which there is insufficient blood flow to the caudal mesoderm causing midline structures to never form in turn, not allowing for precise development of the lower limb.[27] The second theory also involves dysfunction at the caudal mesoderm; however, this theory, the blastogenesis hypothesis, purports that there is a dysfunctional event during gastrulation that causes damage to the caudal mesoderm.[27]

Tethered cord syndrome (TCS) is a rare neurological disorder (related to spina bifida) resulting from congenital defects surrounding gastrulation or injury later in life. Symptoms arise from malformations in tissue attachments that stretch the spinal cord, eventually restricting movement, inducing loss of feeling, or the onset of pain and autonomic symptoms.[28] Errors in gastrulation, neurulation, or primitive streak regression can result in TCS.[29] Specifically, temporary cell-to-cell communication between the yolk sac and external amnion at the neurenteric canal provides critical signaling during normal gastrulation events, which ends upon the completion of successful gastrulation. Continued communication between the yolk and amnion results in persistent progenitor cell proliferation, causing duplication of neuronal tissues (split cord malformation of the vertebral column) or cyst formation (neurenteric cyst).[30]