Developmental Biology - Lecture 3: Embryogenesis Notes

Cleavage and Blastocyst Formation

Cell Potency: Refers to the ability of a cell to differentiate into other cell types.
Greater potency means a cell can differentiate into more cell types.
Lineage potential decreases as cells become differentiated.
Totipotent: Can form any other cell type (e.g., zygote).
Pluripotent: Can form most, but not all, cell types (e.g., inner cell mass of the blastocyst). Cannot form the outer placental component.
Multipotent: Can form a number of different cell types, but they're more restricted (e.g., germ layers: ectoderm, mesoderm, and endoderm).
Unipotent: Can only form one cell type (e.g., erythrocytes or neurons - terminally differentiated).
A progressive restriction of cell fate occurs during development from zygote to differentiated cell types.
Cleavage: Period of rapid cell division following fertilization without an increase in the overall size of the embryo.
The embryo forms increasing numbers of smaller cells called blastomeres.
From the eight-cell stage, cells undergo compaction, becoming tightly joined together via E-cadherin.
The solid ball of cells at this stage is called a morula.
A cavity forms in the morula, resulting in the formation of the blastocyst with inner cell mass and outer trophoblast (or trophectoderm).
At this stage, the embryo starts implanting into the endometrium of the uterus in humans.
Embryonic stem cells (ES cells) derive from the inner cell mass.

Trophoblast vs. Inner Cell Mass Decision

Two hypotheses explain how cells become trophoblast vs. inner cell mass:
- Inside-out hypothesis: Cells in the center become inner cell mass, while those on the outside become trophoblast.
- Cell polarity model: Cells dividing parallel to the zona pellucida become trophoblast, while those dividing perpendicularly become inner cell mass.
The decision to form trophoblast versus inner cell mass depends on the local microenvironment and key transcription factors.
Key transcription factors:
- Oct4: Expression becomes restricted to the inner cells, forming the inner cell mass.
- CDX2: Expressed in the outer cells, forming the trophoblast.
Initially, uniform expression of maternally derived and zygotic Oct4 in the early eight-cell stage embryo.
CDX2 has stochastic (random) expression.
By the morula stage, Oct4 is restricted to inner cells, while CDX2 is restricted to outer cells.
CDX2 represses Oct4 expression, limiting Oct4 to inner cells.
Conversely, Oct4 inhibits CDX2, limiting CDX2 to outer cells.
This dichotomy creates the first molecular transition: inner cell mass expresses Oct4, and the trophoblast expresses CDX2.
Inner cells expressing Oct4 also express Nanog and SOX2.
Oct4, Nanog, and SOX2 are called pluripotency factors and maintain the inner cell mass in a pluripotent state.
Nanog can induce its own expression and activate Sox2 and OCT4.
Immunostaining of a mouse blastocyst shows OCT4 protein in the inner cell mass (green) and CDX2 in the outer trophoblast (red).

Epiblast vs. Hypoblast Decision

The inner cell mass gives rise to the epiblast and the hypoblast.
The entire embryo generates from the epiblast, whereas the hypoblast forms part of the yolk sac.
Commitment to epiblast versus hypoblast may depend on the timing of the cell becoming part of the inner cell mass.
There is initially random (peppered) expression of Nanog and GATA6.
Nanog-expressing cells become epiblast, and GATA6-expressing cells become hypoblast.
Morula (day 4): Solid ball of cells formed via cleavage.
Nanog and GATA6 expression in the inner cell mass at the blastocyst stage resolves those cells into an upper epiblast and a lower hypoblast.
The early blastocyst is a ball of cells with a fluid-filled cavity, inner cell mass.
In the late blastocyst, a bilaminar disc is formed.
The epiblast becomes the entire embryo, and the lower hypoblast forms the yolk sac.
By day 10, the embryo is embedded in the endometrium, and a cavity forms above the epiblast (amnion) and below (yolk sac).
By day 16, the embryo undergoes gastrulation to form the three germ layers.

Gastrulation and Germ Layer Formation

Gastrulation: Formation of the three germ layers: ectoderm, mesoderm, and endoderm.
Cross-section of the embryo shows the bilaminar disc: epiblast (blue) and hypoblast (yellow).
Epiblast cells migrate through the primitive streak, displacing the hypoblast to form the endoderm and then the mesoderm.
The primitive streak forms on the surface of the epiblast.
Cells migrating through the primitive streak displace the hypoblast, forming the definitive endoderm.
A second wave of cells migrates through the primitive streak to form the mesoderm.
Cells that do not migrate through the primitive streak form the ectoderm.
Gastrulation occurs approximately from days 14 through 16 in the human embryo.

Signaling Centers and Anterior-Posterior Axis

Two important signaling centers are set up in the early mammalian embryo:
- Anterior Visceral Endoderm (AVE): A specialized group of cells in the hypoblast.
- Primitive Streak.
The primitive streak forms on the opposite side of the anterior visceral endoderm.
The AVE sends out signals that inhibit the formation of the primitive streak.
In the mouse embryo, the epiblast is a curved layer, and the AVE migrates to the anterior visceral endoderm, sending inhibitory signals to force the primitive streak formation in the epiblast on the opposite side.
Inductive signals set up the primitive streak.
AVE cells set up the anterior-posterior axis and the formation of the primitive streak.
This process involves induction, signaling molecules, and transcription factors.
BMP4 from extraembryonic tissues instructs the epiblast cells to make signaling molecules (Wnt and nodal).
The AVE sends out factors (lefty and cerberus) that are inhibitory to these factors, restricting these factors to one end of the embryo for primitive streak formation.
The primitive streak forms where there are high concentrations of nodal, FGFs, Wnts, the transcription factor brachyury, noggin, chordin, and goosecoid.
A clump of cells forms at the tip of the primitive streak, called the primitive node.
The primitive streak is the site of gastrulation through which cells migrate to form the endoderm or the definitive endoderm, followed subsequently by the mesoderm.
Cells migrating through the node region form the anterior part of the notochord, parts of the neural tube, and head structures.
Cells migrating through the primitive streak further posterior form endoderm and mid- and posterior mesoderm.
The formation of the primitive streak marks the anterior-posterior axis.
Fate mapping in chicken embryos shows cell migration through the primitive streak, with initial cells forming head structures and later cells forming structures in the middle and posterior parts of the body.

Tissue Derivation and Organogenesis

Trophoblast forms the embryonic component of the placenta (cytotrophoblast and syncytiotrophoblast).
The inner cell mass forms a bilaminar disc (epiblast and hypoblast).
Epiblast forms the embryo and the amnion (fluid-filled cavity), and hypoblast forms the yolk sac.
At the implanting blastocyst stage, the bilaminar disc is composed of epiblast and hypoblast cells.
Some cells migrate up and around, forming the amniotic cavity, while the trophoblast forms the cytotrophoblast and the syncytiotrophoblast.
Later, at day 16, the three germ layers form: ectoderm, mesoderm, and endoderm, and mesoderm moves out around the amnion and associates with the trophoblast to form the chorion.
The yolk sac forms the allantois and part of the umbilical cord.
Cytotrophoblast and associated mesoderm become the chorion.
The chorion forms a reticulate pattern of vessel development closely aligned with maternal blood vessels.
Amnion encircles the entire embryo, forming a fluid-filled cushion, and the umbilical cord is formed through blood vessels in the mesoderm and cytotrophoblast.
Regulative development: Early cells of the mammalian embryo show regulative ability, meaning they can produce a normal structure if cells are removed or added.
This capacity is also seen in humans via twinning. Monozygotic twins can form when the early embryo splits (either with two inner cell masses and two trophoblasts, or two inner cell masses sharing one trophoblast).
Conjoined twins can result if the inner cell masses do not fully separate, sharing a common trophoblast.
Dizygotic twins are due to separate fertilization events.
Monzygotic twins are called such because they are derived from one zygote.
The early blastocyst (and differentiation to late blastocyst with epiblast/hypoblast, and gastrulation) results ultimately in a 4 week embryo, with placenta.
Inner cell mass forms either epiblast or hypoblast; the hypoblast forms extra-embyronic endoderm and part of the yolk sac, and will disintergrate due to the importance of the umbilical cord.
The epiblast can form amniotic ectoderm, or alternatively the entire embryo (all embryonic tissues).
Specifically, epiblast can become ectoderm, endoderm, or embryonic mesoderm (and extraembryonic mesoderm).

Derivation of Tissues from Germ Layers

Ectoderm:
- Outer layer of the embryo (demarcated as blue in the lecture).
- Forms the epidermis of the skin and its derivatives (sweat glands, hair follicles).
- Forms the nervous system (brain and central nervous system), sensory systems (parts of the eye), the pituitary gland, jaws, teeth, and neural crest cells.
Mesoderm:
- Middle layer. (demarcated as blue in the lecture)
- Forms the skeleton and muscles, circulatory system, excretory and reproductive system (except germ cells), the dermis of the skin, and the adrenal cortex.
Endoderm:
- Inner layer of the embryo. (demarcated as blue in the lecture)
- Forms the epithelial linings of the digestive tract and associated organs (liver, pancreas).
- Forms the epithelial linings of the respiratory, excretory, and reproductive tracts. Also forms the thymus, thyroid, and parathyroid glands.

Organogenesis

Organogenesis: Differentiation of tissues from the three germ layers and their arrangement into organs.
The three germ layers give rise to four main tissue types:
- Epithelium (endoderm, mesoderm, ectoderm).
- Connective tissue (mesoderm).
- Muscle (mesoderm).
- Nerves (ectoderm).
In the gut, the inner epithelial layer (endoderm), muscle layer (mesoderm), and nerves (ectoderm) have different germ layer sources.

Neurulation

Two key structures form towards the end of gastrulation:
- Notochord (mesodermal origin).
- Neural tube.
The notochord is a solid rod of tissue that supports the embryo, degenerates in mammals, and sends inductive signals to the neighboring ectoderm to form the neural tube.
The neural tube is the future brain and spinal cord, formed from an area of ectoderm called the neural plate, by the process of neurulation. Is also the source of neural crest cells.
Neurulation is marked by the formation of the neural plate at the cranial end of the embryo, growing in a cranial-to-caudal direction.
The lateral edges of the neural plate elevate and move together to form the neural folds, creating a neural groove.
The neural folds fuse together, transforming the neural plate into the neural tube, the precursor to the central nervous system.
During the closure of the neural tube, cells detach from the crest of the neural folds, forming a cell population called the neural crest, which contributes to the peripheral nervous system.
Induction of the neural tube involves the underlying notochord.
The underlying notochord releases sonic hedgehog (SHH), and there are mutually antagonistic actions of dorsalizing and ventralizing factors.
BMPs are strongly expressed in the non-neural ectoderm, with a lower level in the neural ectoderm, and sonic hedgehog is expressed in the notochord.
Sonic hedgehog induces the neural plate by interaction with BMPs.
BMPs become strongly expressed at the dorsal surface, and sonic hedgehog at the ventral surface, creating gradients of BMPs and sonic hedgehog.
Sonic hedgehog is a ventralizing factor, and BMPs are dorsalizing factors.
PAX6 encodes a homeobox transcription factor.
Distal-less homeobox (Dlx) genes are expressed in the ectoderm that will form placodes.
Hox genes are clusters of homeobox genes involved in anterior-posterior patterning.

Lecture Recap

Lecture 1: Principles (induction) and methods (in situ hybridization, immunostaining, western blots) of developmental biology.
Lectures 1 and 2: Regulation of development by transcription factors (Hox genes) and signaling molecules (Wnts, FGFs, receptor tyrosine kinase, TGF beta, Hedgehog).
Lecture 3: Embryogenesis (inner cell mass, blastocyst, sonic hedgehog, growth factors). Covers cell differentiation, gastrulation, and establishment of body axes.