Early Embryonic Development: Morula to Trilaminar Disc

Tight Junctions in the Morula

• Outer morula cells flatten and reorganize their cytoskeleton to create an impermeable seal.
• Tight junctions are built from large trans-membrane proteins that
• adhere two plasma membranes together,
• anchor to the actin cytoskeleton, and
• block paracellular leakage ("sewing" the cells shut).
• Purpose: prevent loss of fluid so the embryo can begin pumping water inward and set up the blastocoel.

Fluid Accumulation & Early Lineage Segregation

• Once sealed, the embryo starts to import fluid; two mechanisms work simultaneously:

1. \text{Na}^+ pumps in trophoblast move sodium into the central cavity → water follows by osmosis.
2. Pinocytosis ("cell drinking"): vesicles internalize extracellular fluid, traverse the cell, and exocytose fluid into the cavity.
   • Cavity expansion physically displaces cells into two populations:
   • Inner Cell Mass (ICM) → future fetus.
   • Trophoblast → future placenta.
   • Despite seeming chaotic, cell movements are highly regulated; each cell "knows" its spatial identity.

Blastocyst Twinning (Early vs. Late Split)

• Early ICM split (before amnion forms) → two embryos, each with its own amniotic sac.
• Late ICM split (after amnion begins) → embryos share a single amniotic sac, allowing cell intermixing (e.g.
absorbed-twin phenomena).
• Both types yield genetically identical (clonal) twins.
• Cause is unclear; probably a mix of genetics & epigenetics—older "zona-tear" theory disproved by live in-vitro imaging.

Osteopontin (OPN) & MMP-9: Molecular Partners

• OPN is secreted by uterine glands at the expected time of embryonic arrival.
• Three key functions (1, 2, 3):
1 Induces blastocyst hatching.
2 Bridges trophoblast & uterine epithelium by binding integrin-type receptors → promotes adhesion.
3 Up-regulates metalloproteinase \text{MMP-9} expression in trophoblast.
• Binding model: OPN (red puzzle piece) fits receptors on both trophoblast & uterine cells, pulling them into nanometer proximity so MMP-9 can act locally.
• MMP-9 digests zona pellucida and then uterine epithelial ECM, creating a controlled “micro-wound.”

Implantation Mechanism & End of Ovum Phase

• Digestion of the uterine surface causes bleeding; tissue trauma releases angiogenic factors → maternal capillaries sprout toward the conceptus.
• Placental trophoblast becomes bathed in maternal blood (esp. in primates) → nutritional shift from histotrophic to hemotrophic supply.
• Successful penetration/vascular anastomosis marks the end of the ovum phase and the start of true embryonic development.
• Analogy: Nude-mouse ear graft—vascularization makes the foreign scaffold a living, integrated tissue, just as maternal vessels invade the implanting embryo.

Germ Layer Formation (Gastrulation) Overview

• Goal: convert the relatively undifferentiated ICM into three primary germ layers—ectoderm, mesoderm, endoderm.
• Happens concurrently with implantation.
• Classic fates:
• Ectoderm → epidermis, nervous system.
• Mesoderm → muscle, bone, heart, body cavities.
• Endoderm → gut tube & associated organs.

Species-Specific Morphogenetic Movements

• Five canonical cell-sheet movements create new layers (species choose one or a combination):

1. Invagination – infolding pocket.
2. Involution – rim rolls inside like a sock.
3. Ingression – individual cells delaminate inward.
4. Delamination – entire sheet splits into two parallel layers (dominant in mammals & food animals).
5. Epiboly – outer cells spread to envelop others (starfish, zebrafish).
   • Metaphors used in lecture:
   • Sock-rolling = involution.
   • Fireworks/shower curtain = epiboly.
   • Finger-trail in cake icing = primitive streak.

Bilaminar Disc Formation (Mammals)

• Delamination yields two sheets:
• Epiblast (upper) – will supply all definitive tissues.
• Hypoblast (lower) – transient yolk-sac lining; template only.
• Space opens above epiblast → amniotic cavity; fluid accumulation creates protective amnion early.
• “Embryonic disc”: flattened, two-layer template that replaces the spherical ICM.

Primitive Streak, Node & Trilaminar Stage

• Primitive streak appears at caudal end and extends cranially, establishing the median axis (right/left, head/tail).
• Primitive node forms at streak tip and functions as an organizer (chemical signaling center).
• Cell migrations:

1. Epiblast cells ingress through streak, displace hypoblast → definitive endoderm.
2. Second wave fills the gap between ectoderm & endoderm → mesoderm.
3. Remaining surface epiblast relabels as ectoderm.
   • Result: trilaminar embryo with germ layers arranged \text{(outside)}\;\text{ectoderm} \;|\; \text{mesoderm} \;|\; \text{endoderm}\;\text{(inside)}.
   • All signaling is autocrine/paracrine; vascular circulation is still establishing.

Clinical, Industrial & Teaching Connections

• Understanding OPN/MMP-9 pathways guides livestock reproductive management and informs human fertility treatments.
• Hormonal manipulations in feed-yard or swine operations ultimately leverage this embryology (timed AI, estrus sync, etc.).
• Veterinary & medical curricula revisit these embryonic fundamentals when diagnosing placental pathologies or congenital defects.
• Real-world analogies (botched surgery re-vascularization; frost-iced severed finger) illustrate the universal importance of blood supply and tissue trauma responses.

• Key timeline markers:
• Tight-junction compaction → morula.
• Fluid influx & cavity formation → early blastocyst.
• OPN-induced hatching → late blastocyst.
• OPN + MMP-9-mediated adhesion/digestion → implantation / end of ovum phase.
• Delamination → bilaminar disc (epiblast + hypoblast).
• Primitive streak/node activity → trilaminar embryo & onset of neurulation (next lecture).