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• ANA301 is Human Embryology taught by Dr. Danielle Bentley, PhD, at the University of Toronto
• The course falls under Unit 1 - Foundations, specifically Topic 3: Implantation and Early Intra-Embryonic Development

T3 Learning Outcomes

• List and explain the sequential changes in blastocyst formation from a zygote
• Summarize extra-embryonic events of implantation
• Summarize intra-embryonic events of bilaminar disk formation
• Describe cell movements during gastrulation
  • Distinguish and compare the epiblast from the ectoderm
  • Distinguish and compare the hypoblast from the endoderm
• List and describe each step in notochord formation
• Describe sequential cell movements during neurulation
• Align gastrulation, notochord formation, and neurulation events to specific dates

Intra-embryonic Development Timeline

• Week One: The zygote travels from the site of fertilization to the implantation site and cleaves
• Week Two: Implantation, with bilaminar disk and cavity formation
• Week Three: Gastrulation and early neurulation

Week One: Cleavage of The Zygote

• Mitotic divisions produce blastomeres
• Each blastomere is totipotent, with the ability to develop into intra-embryonic and extra-embryonic structures
• Blastomeres travel within the Zona Pellucida (ZP) along the uterine tube towards the uterine cavity
• Compaction begins after the 8-cell stage

Week One: Compaction

• Starts after the 8-cell stage, between the 3rd and 4th cleavage
• Blastomeres "compact" and adhere to form a tightly packed ball
• Outer cells flatten and surround the inner cells

Week One: Compaction Results

• Trophoblast(outer cell mass) will form extra-embryonic tissues like the placenta, and does not contribute to the embryo proper
• Embryoblast (inner cell mass) will form the intra-embryonic tissues, amniotic membrane, and the lining of the primitive umbilical vesicle

Week One: Formation of the Blastocyst

• Around day 3, the morula enters the uterine cavity
• Around day 4, fluid enters the morula, creating the blastocyst cavity (blastocoele)
• The conceptus is now called a blastocyst
• The cavity displaces the embryoblast to the embryonic pole
• The blastocyst is still contained within the degenerating zona pellucida
• Between days 4 and 6, the blastocyst hatches
• This is facilitated by proteolytic enzymes from the trophoblast, which degenerate the zona pellucida

Week Two: Implantation

• Blastocyst implantation events occur between day 6 and day 10
• On day 6, the trophoblast overlying the embryonic pole contacts the uterine mucosa
• The blastocyst is captured from the uterine cavity by the uterine epithelium

Week Two: Trophoblast Cells

• On day 7, trophoblast cells next to the uterine mucosa proliferate and penetrate uterine tissue
• They fuse to form a multinucleated mass of cytoplasm called the syncytiotrophoblast
• Remaining trophoblast, the cytotrophoblast, is mitotically active
• Newly formed cells either remain in the cytotrophoblast OR migrate into syncytiotrophoblast

Week Two: Syncytiotrophoblast

• On day 8, continues to erode uterine tissue through proteolytic enzymes, enabling the blastocyst to burrow
• Erodes local uterine blood vessels upon contact
• Produces Human Chorionic Gonadotropin (hCG), which is absorbed into maternal blood and excreted in maternal urine

Week Two: Vacuoles

• On day 9, vacuoles appear in the syncytium
• Vacuoles will fuse to form lacunae
• This is called the lacunar stage of trophoblast development

Week Two: Blastocyst Implantation

• On day 10, the blastocyst embeds deep into uterine tissue = official "implanted"
• The surface defect in the endometrial epithelium is preliminarily closed by a closing plug (fibrin coagulum of blood) Days 10 and 11: Adjacent lacunae fuse, forming lacunar networks, filled with embryotroph (blood + gland cellular debris)
• Days 11 and 12: Maternal blood from eroded endometrial blood vessels flows through lacunar networks, initiating uteroplacental "circulation"
• Days 12 and 13: Defect in endometrial endothelium is repaired

Week Two: Ovarian and Menstrual Cycle

• Around day 21 (gestational age), first contact occurs
• Around day 27 (gestational age), endometrial endothelium is repaired

Week Two: Clinical Cases

• Ectopic Pregnancy: When the embryo implants outside the uterine cavity
• Incidence: 1 in 100 pregnancies, >95% are in the uterine tube.
• Tubal Pregnancy: Hatched blastocyst implants in the ampulla or isthmus of the uterine tube
  • Typically caused by uterine tube pathologies which delay tubat transit time
  • Risk of rupture and life-threatening maternal haemorrhage
  • Treatment: surgical removal

Week Two: Bilaminar Embryonic Disc

• A flat, two-layered disk will ultimately become all intra-embryonic tissues
• Formation begins as the blastocyst implants, between days 7 and 14
• Once complete, the bilaminar embryonic disk will have two fluid-filled spaces on either side of it

Week Two: Embryoblast Differentiation

• On day 8, While the trophoblast layers are working on implantation, the embryoblast (inner cell mass) differentiates into two layers
• Hypoblast Layer: Small cuboidal cells adjacent to the exocoelomic cavity
  • Migrating hypoblast cells form the exocoelomic membrane adjacent to the cells of the cytotrophoblast)
  • The exocoelomic cavity is lined by the exocoelomic membrane and the hypoblast
• Epiblast Layer: high columnar cells adjacent to the amniotic cavity
  • Migrating epiblast cells separate form the amnion (adjacent to the cells of the cytotrophoblast)
  • The amniotic cavity is lined by the amnion and the epiblast
• On day 9, a fine, loose connective tissue first appears coming from from the exocoelomic membrane, and is called extraembryonic mesoderm
  • It surrounds the amnion and the exocoelomic cavity/primary umbilical vesicle
• On day 12, cavities form within the extraembryonic mesoderm, called extraembryonic coelomic spaces

Week Two: Blastocyst Protrusion and Naming

• By day 13, embedded blastocyst slightly protrudes into the uterine lumen as the endometrial defect heals Extraembryonic coelomic spaces fuse, creating the extraembryonic coelom (chorionic cavity)
  • The Extraembryonic coelom/chorionic cavity surrounds the amnion and the primary umbilical vesicle EXCEPT at the connecting stalk
• The Primary umbilical vesicle decreases in size, which changes its name to the secondary umbilical vesicle
• The extraembryonic mesoderm is now named according to location
  • Extraembryonic somatic mesoderm:Adjacent to cytotrophoblast and covers the amnion
  • Extraembryonic splanchnic mesoderm: covers the umbilical vesicle

Week Two: Intra-Embryonic Cells Shape

• By day 14, known pregnancy status usually determined
• As the extraembryonic coelom squishes the primary umbilical vesicle into the secondary umbilical vesicle, a portion of the primary vesicle may persist as an exocoelomic cyst
• Intra-embryonic cells are still the shape of a bilaminar disk, though hypoblast at the cranial end become tall columnar cells, forming the prechordal plate, which is the future site of the mouth

Week Three: Gastrulation

• During the third week, gastrulation occurs, which is the transformation of the gastrula from a bilaminar disk to a trilaminar disk
• Begins with primitive streak formation at the caudal end of the epiblast
• Clearly visible by day 15/16
• Sets up each axis of the body
• The cranial tip of the primitive streak is the primitive node, a slightly elevated area surrounding a small primitive pit
• Epiblast cells migrate toward the primitive streak, detach from epiblast, and slip inwards
• Dynamic cell migration process results in three germ cell layers

Week Three: Germ Layers

• Some invaginating/migrating cells displace hypoblast cells and form the endoderm
• Most invaginating/migrating cells end up between endoderm, epiblast and form mesoderm
• Non-migrating epiblast cells form the ectoderm

Week Three: Active Primitive Streak

• Cells invaginate during gastrulation, and proliferate and migrate throughout the embryonic disk
• Cells that invaginate through the primitive node migrate cranially along the midline
• The primitive streak is active until early in the 4th week.
  • Disappears by the end of the 4th week (day 28)

Week Three: Notochord Formation

• Notochord resembles a rigid, rod-like structure that stretches along the longitudinal axis of the embryo
• General functions: Provides rigidity and support to the embryonic disk
• Serves as the primary inductor (signaling center) in the early embryo, and induces neurulation
• Provides signals for future musculoskeletal development
• Indicates the future site of vertebral bodies, and assists in defining embryo axis
• The notochord degenerates as the bodies of the vertebrae are formed
  • Small portions persist as the nucleus pulposus of each intervertebral disc

Week Three: Notochordal Process

• Epiblast cells that invaginate through the primitive node migrate cranially along the midline
• The cells form a cord-like structure, the notochordal process
• It extends between the primitive node and the prechordal plate
• The prechordal plate has columnar endodermal cells at the cranial end of the embryo where ectoderm and endoderm are fused (no mesoderm in between)
• The plate contributes endodermal cells to the oropharyngeal membrane (future site of the oral cavity)
• The notochordal process soon acquires a lumen to form the: notochordal canal

Week Three: Transformation of Notochordal Process

• The floor of the notochordal process fuses with the underlying embryonic endoderm
  • Fused cells degenerate, creating holes that grow/merge as the floor disappears
  • Notochordal canal is obliterated
• Amniotic cavity (dorsally) is now in direct contact with the umbilical vesicle (ventrally) via the neurenteric canal
• The roof of the notochordal process becomes the notochordal plate
• Beginning at the cranial end, cells at the lateral edges of the notochordal plate proliferate and bilaterally fold ventrally, and fusion creates the notochord
• Endoderm fills in ventrally, reestablishing the continuous cellular layer

Neurulation

• Represents the beginning of nervous system development
• It starts as the notochord develops and is completed by the end of week 4
• The notochord induces the overlying ectoderm to thicken into the neural plate
• Around day 18, the lateral edges of the neural plate (called neural folds) elevate dorsally
  • The tip of each neural fold contains neural crest cells which will remain separate from the neural tube
  • The space in between the two neural folds is the neural groove
• The bilateral neural folds continue to elevate and bend medially, and toward each other, fusion creates the neural tube, which separates the:
  • Neuroectoderm (green)
  • Surface Ectoderm (blue/grey)
• Neurulation results in the separation of the neuroectoderm from the surface ectoderm
  • Neuroectoderm becomes the neural tube
    • Neural crest cells migrate within the mesoderm, and come from the tips of the neural folds
  • The surface ectoderm becomes the epidermis of the skin

Clinical Cases: Neural Tube Defects

• Can result from disruption to neurulation, typically at a neuropore
• Failure of the cranial neuropore: meroencephaly to anencephaly
  • Lethal defect, detected early in pregnancy when imaging is available
  • Neural tissue is exposed to amniotic fluid and degraded