TARGET DECK: MED::I::Morphology and Development::Embryology::03 - The Second Week of Human Development
Embryology — Lesson 3
Embryoblast Formation
The inner cells of the morula — the embryoblast (or inner cell mass, ICM) — are surrounded by a layer of flattened blastomeres that form the trophoblast. Hippo signaling is an essential factor in segregating the ICM from the trophoblast.
Early Pregnancy Factor
An immunosuppressant protein — the early pregnancy factor — is secreted by trophoblastic cells and appears in the maternal serum within 24 to 48 hours after implantation. It forms the basis for a pregnancy test applicable during the first 10 days of development.
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The early pregnancy factor appears in maternal serum within {1:24 to 48 hours} after implantation and is secreted by {2:trophoblastic cells}.
Gametogenesis — Formation of the Blastocyst
Shortly after the morula enters the uterus (~4 days after fertilization), uterine fluid passes through the zona pellucida to form a fluid-filled space — the blastocystic cavity — inside the morula.
As fluid increases in the cavity, the blastomeres are separated into two parts:
| Structure | Origin | Function |
|---|---|---|
| Trophoblast | Outer, thin cells | Gives rise to the embryonic part of the placenta |
| Embryoblast (ICM) | Discrete inner group | Primordium of the embryo |
Beginning at the 8-Cell Stage
Compaction
At the 8-cell stage:
- Intercellular adhesions are initially weak and each blastomere is morphologically recognizable
- The first event is an increase in E-cadherin, causing cell boundaries to become less evident → COMPACTION
Polarity Establishment
In parallel with compaction, blastomeres undergo intracellular polarization, resulting in the asymmetric distribution of defined cellular components between the apical and basolateral membrane domains, establishing a radial apical–basolateral axis of cell polarity.
- Apical domain: PAR-6 / aPKC / Cdc42, with tight junctions (ZO-1, Claudin)
- Basolateral domain: PAR-1 / Scribble / Lgl, with adherens junctions (E-cadherin, catenins)
Developmental Potential at 4- and 8-Cell Stage
Although individual cells separated from the 4- or 8-cell stage mouse embryo cannot independently develop beyond implantation, they are nevertheless able to contribute to all tissues when combined with other blastomeres in experimentally derived chimaeras — indicating they retain full developmental potential.
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At the 8-cell stage, cell boundaries become less evident due to an increase in {1:E-cadherin}, a process known as {2:compaction}.
Tight Junction Formation and First Cell-Fate Decision
First Cell-Fate Decision
Tight junction formation is initiated at the border between the apical and basolateral regions, delineating the two membrane domains.
The initial differences in cell position (outer/inner) and intracellular organization (polarized/nonpolarized) will eventually segregate blastomeres into one of two lineages:
- Trophectoderm (TE)
- Inner cell mass (ICM)
This is the first cell-fate decision.
Models of First Cell-Fate Decision
Polarity model: Differences arise from asymmetric partitioning of polarized subcellular components between daughter cells upon asymmetric cell division (differential inheritance of apical vs. basolateral membrane domains).
Positional (inside-outside) model: Differences arise from the differential extent of cell-to-cell contact corresponding to each blastomere’s relative position in the embryo.
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The first cell-fate decision segregates blastomeres into {1:trophectoderm (TE)} and {2:inner cell mass (ICM)}.
At Morula Stage (>16 cells)
The mouse embryo develops into a cyst-like structure:
- Outer cells remain polarized → express Cdx2, Gata3 → become TE
- Inner cells lose polarity → express Sox2, Oct3/4, Nanog → become ICM
Plasticity at 16-Cell Stage
At the 16-cell stage, inner cell mass cells transplanted to the outer surface of another embryo can become trophoblast, and at least some outer cells can turn into ICM if transplanted into the interior.
By the 32-cell stage, this capacity for phenotypic transformation is largely lost.
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By the {1:32-cell stage}, the capacity for phenotypic transformation between TE and ICM is largely lost.
Positional Inside-Outside Hypothesis
Classic Experiment
- If a marked blastomere is inserted into the interior of a morula → it and its progeny become part of the inner cell mass
- If a marked blastomere is placed on the outside of a host morula → it and its descendants contribute to the trophoblast
Hippo Signalling Pathway
Hippo Pathway in Cell-Fate Determination
- Inner cells: Hippo pathway is active → Lats1/2 kinases are activated → Yap1 is phosphorylated and sequestered in the cytoplasm → Tead4-dependent transcription of TE genes is blocked
- Outer cells: Hippo pathway is suppressed → Lats1/2 are not activated → unphosphorylated Yap1 enters the nucleus → Tead4-dependent transcription of TE genes is enabled
Result: Only outer cells can express trophectoderm genes.
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In outer cells, failure to activate {1:Lats1/2} kinases permits unphosphorylated {2:Yap1} to enter the nucleus and enables {3:Tead4}-dependent transcription of trophectoderm-required genes.
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In inner cells, activation of Lats1/2 leads to {1:phosphorylation and cytoplasmic sequestration} of Yap1, preventing {2:trophectoderm} gene transcription.
Parental Imprinting
Parental Imprinting
Paternal genes are predominantly expressed in the trophoblast.
Called parental imprinting, its effects are demonstrated by pronuclear transplant experiments:
Zygote composition Embryo Placenta & Yolk Sac Two female pronuclei Develops fairly normally Poorly developed Two male pronuclei Severely stunted Nearly normal Normal (one of each) Normal Normal Parental imprinting occurs during gametogenesis.
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A zygote with two {1:female} pronuclei has a nearly normal embryo but poorly developed placenta and yolk sac, while two {2:male} pronuclei produce a stunted embryo with a nearly normal placenta, demonstrating {3:parental imprinting}.
Twinning: Monozygotic Twins
Modes of Monozygotic Twinning
- A: Cleavage of the early embryo → each half develops as a completely separate embryo
- B: Splitting of the inner cell mass → two embryos enclosed within a common trophoblast (most common mode)
- C: Inner cell mass does not completely separate → may result in conjoined twins
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The most common mode of monozygotic twinning involves splitting of the {1:inner cell mass}, resulting in two embryos enclosed within a common {2:trophoblast}.
Zona Pellucida — Summary of Functions
Functions of the Zona Pellucida
- Promotes maturation of the oocyte and follicle
- Acts as a species-specific barrier, normally allowing only sperm of the same species access to the egg
- Initiates the acrosomal reaction
- After fertilization, prevents additional spermatozoa from reaching the zygote (polyspermy block)
- During early cleavage, acts as a porous filter for uterine tube secretions
- Lacks histocompatibility (HLA) antigens → serves as an immunological barrier between mother and embryo
- Prevents blastomeres of the early cleaving embryo from dissociating
- Facilitates differentiation of trophoblastic cells
- Normally prevents premature implantation into the wall of the uterine tube
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The zona pellucida lacks {1:histocompatibility (HLA) antigens}, serving as an {2:immunological barrier} between the mother and the antigenically different embryo.
Day 5: Blastocyst Hatching
At Day 5, the blastocyst hatches from the zona pellucida.
End of First Week: Initiating Implantation
Uterine Preparation for Implantation
- Very soon after arriving in the uterus, the blastocyst becomes tightly adherent to the uterine lining
- Endometrial stromal cells respond to the blastocyst and to progesterone (from the corpus luteum) by differentiating into decidual cells → decidual reaction
- Endometrial glands enlarge; local uterine wall becomes highly vascularized
- Secretions of decidual cells and endometrial glands include growth factors and metabolites that support embryo growth
hCG and Corpus Luteum Maintenance
If an embryo implants, trophoblastic cells produce human chorionic gonadotropin (hCG), which:
- Supports the corpus luteum
- Maintains the supply of progesterone (maternal recognition of pregnancy)
Ectopic Pregnancy
Occasionally, a blastocyst implants in an abnormal site:
- Peritoneal cavity
- Surface of the ovary
- Within the oviduct
- Abnormal site within the uterus
Ectopic pregnancies often threaten the life of the mother because blood vessels at the abnormal site may rupture as the embryo and placenta grow. Symptoms include abdominal pain and/or vaginal bleeding.
Intervention: methotrexate (blocks rapid cell division) or surgical intervention.
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hCG is produced by the {1:trophoblast} and maintains the {2:corpus luteum}, thereby sustaining {3:progesterone} secretion during early pregnancy.
Initiating Implantation — Day 6
Approximately 6 days after fertilization, the blastocyst attaches to the endometrial epithelium.
Attachment Polarity
Attachment occurs at the area above the inner cell mass (embryonic pole), indicating that trophoblast surface properties are not uniform.
As soon as the blastocyst attaches, the trophoblast differentiates into two layers:
| Layer | Description |
|---|---|
| Cytotrophoblast | Inner layer of mononucleated cells; mitotically active |
| Syncytiotrophoblast | Outer layer; multinucleate protoplasmic mass formed by cell fusion; no discernible cell boundaries |
Invasion of the Endometrium
Finger-like processes of the syncytiotrophoblast extend through the endometrial epithelium and invade the endometrial connective tissue. By the end of the first week, the blastocyst is superficially implanted in the compact layer.
Syncytiotrophoblast Actions
- Produces proteolytic enzymes that erode maternal tissues, enabling the blastocyst to “burrow” into the endometrium
- Decidual cells help control the depth of penetration
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The syncytiotrophoblast is a {1:multinucleate} protoplasmic mass formed by {2:fusion} of cytotrophoblastic cells, and produces {3:proteolytic enzymes} to invade the endometrium.
At the end of the first week, a cuboidal layer of cells — the hypoblast — appears on the surface of the embryoblast, facing the blastocystic cavity.
Decidual Reaction
Decidual Reaction
While the embryo burrows into the endometrium, fibroblast-like stromal cells of the edematous endometrium swell with accumulation of glycogen and lipid droplets → these are called decidual cells.
Decidual cells:
- Are tightly adherent and form a massive cellular matrix
- First surround the implanting embryo, then occupy most of the endometrium
Concurrently, infiltrating leukocytes in the endometrial stroma secrete interleukin-2, which prevents maternal recognition of the embryo as a foreign body during early implantation.
Primary function: provide an immunologically privileged site to protect the developing embryo from rejection.
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During the decidual reaction, endometrial stromal cells accumulate {1:glycogen and lipid droplets} and are called {2:decidual cells}; leukocytes concurrently secrete {3:interleukin-2} to prevent maternal immune recognition.
Why Isn’t the Conceptus Rejected by Its Mother?
Medawar's Three Hypotheses (Nobel Prize 1960)
- Fetal and maternal cells are physically separated from one another
- The conceptus is antigenically immature
- The maternal immune system is suppressed or becomes tolerant to the conceptus during pregnancy
Current Understanding
- The trophoblast separates fetal tissues from maternal tissues and poorly expresses MHC molecules (antigenically immature)
- Evidence shows maternal T cells are activated during pregnancy
- Tolerogenic mechanisms block maternal T-cell responses and prevent fetal rejection
- The unique hormonal conditions of pregnancy induce specific tolerance to fetal antigens
- Maternal antiviral immunity is not suppressed during pregnancy (e.g., HIV+ women do not suffer AIDS-like disease during pregnancy)
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The trophoblast poorly expresses {1:MHC molecules}, contributing to immune tolerance; tolerance during pregnancy is {2:specific} to fetal antigens and does not suppress {3:antiviral} immunity.
Bilaminar Embryonic Disc — Second Week
Formation of the Bilaminar Disc
Implantation is completed during the second week with formation of the bilaminar embryonic disc, composed of:
- Epiblast (dorsal) — columnar cells
- Hypoblast / primitive endoderm (ventral) — cuboidal cells
An extracellular basement membrane is laid down between the two layers.
The embryonic disc gives rise to all germ layers that form the tissues and organs of the embryo.
Extraembryonic structures forming during the second week:
- Amniotic cavity
- Amnion
- Umbilical vesicle (yolk sac)
- Connecting stalk
- Chorionic sac
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The bilaminar embryonic disc consists of the {1:epiblast} (dorsal) and {2:hypoblast} (ventral), with the {3:epiblast} believed to contain all cells that will generate the actual embryo.
Implantation — Detailed Progression
Syncytiotrophoblast Expansion
Syncytiotrophoblastic cells displace endometrial cells via proteolytic enzymes; endometrial cells undergo apoptosis to facilitate implantation. Uterine connective tissue cells accumulate glycogen and lipids, forming decidual cells.
Cytotrophoblast Role
The cytotrophoblast is mitotically active: it forms new trophoblastic cells that migrate into and fuse with the syncytiotrophoblast, losing their cell membranes.
hCG Production
- The syncytiotrophoblast produces hCG, which enters maternal blood through lacunae in the syncytiotrophoblast
- hCG maintains development of spiral arteries in the myometrium and formation of the syncytiotrophoblast
- hCG forms the basis for pregnancy tests; enough is produced by the end of the second week to give a positive result
Day 9 — Coagulation Plug
By day nine, the syncytiotrophoblast blankets the entire blastocyst. A plug of acellular material — the coagulation plug — seals the small implantation hole in the endometrial epithelium.
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By day {1:9}, the syncytiotrophoblast surrounds the entire blastocyst; a {2:coagulation plug} temporarily seals the implantation site in the endometrial epithelium.
Regulation of Blastocyst Adherence to the Uterine Epithelium
Hormonal Control of Uterine Receptivity
The uterus cycles through receptive and non-receptive stages, controlled by estrogen and progesterone:
- Estrogen (via estrogen receptor): stimulates endometrial proliferation by inducing production of growth factors (e.g., IGF-1); prevents programmed cell death in the uterine epithelium
- Progesterone (via progesterone receptor): blocks continued endometrial growth and allows implantation to occur
Receptive Stage Changes
During the receptive stage:
- Apical glycocalyx (including high-molecular-weight mucin glycoproteins) decreases in amount
- Apical microvilli retract, establishing a flattened surface
- Large apical protrusions called pinopodes form
Blastocyst Attachment Factors
- The zona pellucida prevents premature attachment; even after early zona removal the blastocyst itself remains in an attachment-incompetent stage
- Maturing blastocysts express perlecan (a heparan sulfate proteoglycan) on their surface
- Heparan sulfate proteoglycans bind extracellular matrix proteins and growth factors/cytokines → serve as attachment factors
- The uterus upregulates Hb-EGF (heparin-binding EGF-like growth factor) at implantation sites; Hb-EGF binding requires the blastocyst to express both EGF receptor and heparan sulfate proteoglycan
- Perlecan-null mice show no implantation defects, suggesting functional redundancy among heparan sulfate proteoglycans
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During the receptive stage of the uterus, apical {1:glycocalyx} decreases, microvilli retract, and large protrusions called {2:pinopodes} form on the endometrial epithelium.
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Hb-EGF binding to the blastocyst requires expression of both the {1:EGF receptor} and {2:heparan sulfate proteoglycan} on the blastocyst surface.
Embryoblast Reorganizes into Epiblast and Hypoblast — Day 8
By day 8, the embryoblast consists of:
- Epiblast: external/upper layer of columnar cells (dorsal)
- Hypoblast: internal/lower layer of cuboidal cells (ventral)
An extracellular basement membrane is laid down between the two layers → forming the bilaminar embryonic disc (bilaminar blastoderm).
Dorsal–Ventral Axis
Formation of the bilaminar embryonic disc defines the primitive dorsal–ventral axis:
- Epiblast = dorsal
- Hypoblast = ventral
Amniotic Cavity — Day 8
The first new cavity of the second week — the amniotic cavity — appears on day 8 as fluid collects between cells of the epiblast and the overlying trophoblast.
- A layer of epiblast cells expands toward the embryonic pole and differentiates into a thin amnion membrane, separating the new cavity from the cytotrophoblast
- The amnion is one of four extraembryonic membranes: amnion, chorion, yolk sac, and allantois
- The amniotic cavity fills with amniotic fluid, which acts as a shock absorber and prevents desiccation of the developing embryo
- By the eighth week, the amnion encloses the entire embryo
Important
The embryonic epiblast is believed to contain all the cells that will generate the actual embryo.
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The amniotic cavity forms on day {1:8} as fluid collects between the {2:epiblast} and the overlying trophoblast; it is lined by the {3:amnion}.
Development of Yolk Sac and Chorionic Cavity
Primary Yolk Sac (Heuser’s Membrane) — Day 8
Proliferation of hypoblast cells, followed by two successive waves of cell migration, forms the yolk sac membranes:
- First wave (day 8): forms the primary yolk sac = exocoelomic membrane = Heuser’s membrane, which extends from the hypoblast into the blastocyst cavity
Extraembryonic Mesoderm Formation
Simultaneously, extraembryonic mesoderm forms, filling the blastocyst cavity with loosely arranged cells (believed to originate from the hypoblast in humans).
Role of Extraembryonic Mesoderm
- Joins the trophoblastic extension → gives rise to blood vessels that carry nutrients from mother to embryo
- The narrow connecting stalk of extraembryonic mesoderm that links the embryo to the trophoblast eventually forms the vessels of the umbilical cord
- Trophoblast tissue + blood vessel-containing mesoderm = chorion → fuses with uterine wall → placenta
- Maternal portion: uterine endometrium (modified during pregnancy)
- Fetal component: chorion
Chorionic Cavity — Days 10–13
The extraembryonic coelom (chorionic cavity) is formed by splitting of the extraembryonic mesoderm into two layers:
- Extraembryonic somatic mesoderm (chorionic plate): lines the cytotrophoblast and amnion
- Extraembryonic splanchnic mesoderm: covers the yolk sac
By day 13, the embryonic disc with its dorsal amnion and yolk sac is suspended in the chorionic cavity solely by the connecting stalk.
Secondary Yolk Sac — Day 12
By day 12, the primary yolk sac is displaced (and eventually degenerates) by the second wave of migrating hypoblast cells → forms the secondary yolk sac.
Functions of the Definitive Yolk Sac
- Remains a major structure through the fourth week
- Extraembryonic mesoderm of the outer yolk sac layer is a major site of hematopoiesis (blood formation)
- After the fourth week, the yolk sac is rapidly overgrown by the developing embryonic disc
- Normally disappears before birth; persists in rare cases as Meckel’s diverticulum
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The primary yolk sac is formed by the {1:first} wave of hypoblast cell migration on day {2:8}, while the secondary yolk sac is formed by the {3:second} wave on day {4:12}.
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The extraembryonic mesoderm of the outer yolk sac is a major early site of {1:hematopoiesis}; persistence of the yolk sac after birth may result in {2:Meckel’s diverticulum}.
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The connecting stalk of {1:extraembryonic mesoderm} eventually forms the {2:vessels of the umbilical cord}.
Timeline of the Second Week
| Days | Key Events |
|---|---|
| ~7–8 | Trophoblast differentiates into cytotrophoblast and syncytiotrophoblast; implantation begins; bilaminar disc forms |
| ~8 | Amniotic cavity forms; syncytiotrophoblast expands |
| ~8–9 | Cells migrate from hypoblast to form primary yolk sac; lacunae form within trophoblast; implantation complete; syncytiotrophoblast surrounds embryo |
| ~10–11 | Extraembryonic mesoderm forms and splits to form chorionic cavity; trophoblastic lacunae anastomose with maternal blood sinusoids |
| ~12 | Cells migrate from hypoblast to form secondary yolk sac; primary yolk sac pushed aside and begins to degenerate |
| ~13 | Primary yolk sac reduced to remnant at abembryonic pole of chorionic cavity; embryo attached to chorion by connecting stalk |
https://studentconsult.inkling.com/read/moore-before-we-are-born-9/animations/implantation
Uteroplacental Circulatory System — Development During Second Week
From Diffusion to Active Circulation
During the first week, the embryo obtains nutrients and eliminates wastes by simple diffusion. Rapid growth makes a more efficient system necessary → uteroplacental circulation develops.
Day 9: Trophoblastic Lacunae
- Vacuoles called trophoblastic lacunae open within the syncytiotrophoblast
- Maternal capillaries expand to form maternal sinusoids that rapidly anastomose with the trophoblastic lacunae
Days 11–13: Primary Chorionic Stem Villi
- Anastomoses continue to develop
- Cytotrophoblast proliferates locally, forming extensions that grow into the overlying syncytiotrophoblast → primary chorionic stem villi
- Growth is believed to be induced by the underlying extraembryonic somatic mesoderm
Gas and Nutrient Exchange
- Oxygenated blood enters lacunae from spiral endometrial arteries
- Deoxygenated blood is removed through endometrial veins
- The endothelial lining of maternal sinusoids does not invade the trophoblastic lacunae → no maternal layer needs to be crossed in the diffusion barrier
Day 16: Secondary Chorionic Stem Villi
Extraembryonic mesoderm penetrates the core of primary stem villi → secondary chorionic stem villi
End of Third Week: Tertiary Chorionic Stem Villi
Villous mesoderm gives rise to blood vessels that connect with vessels forming in the embryo proper → working uteroplacental circulation established. The primitive heart starts beating on day 22.
Diffusion Barrier Across the Villus
Gases, nutrients, and wastes must cross four tissue layers:
| Layer | Tissue |
|---|---|
| 1 | Endothelium of villus capillaries |
| 2 | Loose connective tissue (extraembryonic mesoderm) in the villus core |
| 3 | Cytotrophoblast |
| 4 | Syncytiotrophoblast |
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Trophoblastic lacunae appear within the syncytiotrophoblast on day {1:9}; cytotrophoblastic extensions that grow into these lacunae are called {2:primary chorionic stem villi}.
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The four tissue layers that gases and nutrients must cross in the uteroplacental diffusion barrier are: {1:endothelium of villus capillaries}, {2:loose connective tissue (extraembryonic mesoderm)}, {3:cytotrophoblast}, and {4:syncytiotrophoblast}.
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The primitive heart starts beating on day {1:22}, coinciding with the establishment of a working {2:uteroplacental circulation} at the end of the third week.
https://studentconsult.inkling.com/read/larsen-human-embryology-schoenwolf-5/videos/animation-2-2
The Rule of Twos — Mnemonic for the Second Week
Rule of Twos
Many events during the second week occur in pairs:
Structure/Event Two Products Embryoblast Epiblast + Hypoblast Trophoblast Cytotrophoblast + Syncytiotrophoblast Yolk sacs Primary + Secondary New cavities Amniotic cavity + Chorionic cavity Extraembryonic mesoderm layers Somatic (chorionic plate) + Splanchnic (yolk sac covering)
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During the second week (Rule of Twos): the embryoblast splits into {1:epiblast and hypoblast}; the trophoblast gives rise to {2:cytotrophoblast and syncytiotrophoblast}; two yolk sacs form ({3:primary and secondary}); two new cavities form ({4:amniotic and chorionic cavities}).
TLDR
Lesson 3 — Key Points
- Blastocyst formation (~day 4): morula enters uterus; uterine fluid forms blastocystic cavity; blastomeres segregate into trophoblast (outer) and embryoblast/ICM (inner)
- Compaction (8-cell stage): E-cadherin increases; cell boundaries become less evident; tight junctions form at apical–basolateral boundary; blastomeres polarize
- First cell-fate decision: outer (polarized) cells → trophectoderm (Cdx2, Gata3); inner (apolar) cells → ICM (Oct3/4, Nanog, Sox2). Explained by polarity and positional (inside-outside) models
- Hippo pathway: active in inner cells → Lats1/2 phosphorylate Yap1 → cytoplasmic sequestration → TE gene transcription blocked; inactive in outer cells → Yap1 nuclear entry → Tead4-driven TE gene expression
- Parental imprinting: paternal genes dominate in trophoblast; two maternal pronuclei → near-normal embryo but poor placenta; two paternal pronuclei → stunted embryo but near-normal placenta
- Monozygotic twinning: arises from cleavage of early embryo, splitting of ICM (most common), or incomplete ICM separation (conjoined twins)
- Zona pellucida: species barrier, acrosome reaction initiator, polyspermy block, immunological shield, prevents premature implantation
- Day 5: blastocyst hatches; Day 6: attaches to endometrium at embryonic pole; trophoblast → cytotrophoblast + syncytiotrophoblast
- Decidual reaction: stromal cells → decidual cells (glycogen + lipid droplets); IL-2 from leukocytes prevents early maternal immune rejection
- Medawar’s hypotheses for immune tolerance: physical separation, antigenic immaturity, maternal immune suppression. Trophoblast poorly expresses MHC; specific tolerance; antiviral immunity preserved
- Day 8: bilaminar disc (epiblast dorsal, hypoblast ventral); amniotic cavity forms; Day 9: coagulation plug; trophoblastic lacunae open
- Yolk sacs: primary (Heuser’s membrane, day 8) → secondary (day 12, replaces primary); extraembryonic mesoderm lines outer yolk sac → hematopoiesis; remnant persistence = Meckel’s diverticulum
- Extraembryonic mesoderm splits into somatic (chorionic plate + amnion lining) and splanchnic (yolk sac covering) layers → chorionic cavity
- Connecting stalk: extraembryonic mesoderm bridging embryo to trophoblast → future umbilical cord vessels
- Uteroplacental circulation: lacunae (day 9) → anastomose with maternal sinusoids → primary villi (days 11–13) → secondary villi (day 16, mesoderm core) → tertiary villi (end of week 3, vascularized) → heart beats day 22
- Diffusion barrier (4 layers): villus capillary endothelium → loose CT (extraembryonic mesoderm) → cytotrophoblast → syncytiotrophoblast
- hCG: produced by syncytiotrophoblast; maintains corpus luteum → progesterone; positive pregnancy test by end of week 2
- Rule of Twos: epiblast/hypoblast; cytotrophoblast/syncytiotrophoblast; primary/secondary yolk sac; amniotic/chorionic cavities; somatic/splanchnic extraembryonic mesoderm