Developmental Biology Lecture 7
Early Development of Fish, Birds, and Mammals
Each undergoes meroblastic cleavage, characterized by:
A small portion of the embryo dividing while
Most of the yolk remains undivided.
Early Development in Fish
Model Organism: Teleost Danio rerio (zebrafish)
Convenient system due to:
They breed all year round.
Development occurs externally.
Embryos are transparent, facilitating observation.
They are easy to maintain.
They develop rapidly, reaching a free-swimming larval stage with functional organs within 5 days.
Large Scale Mutagenesis Screen
Zebrafish became the first vertebrate screened for developmental mutants.
Procedure:
Males were exposed to a chemical mutagen.
The following generations can be described as:
Parents:
Mutagenized: +/m
Wild-type: +/+
F₁ Generation:
Types: +/m, +/+
F₂ Generation:
Types: +/m, +/+
Results Table for Phenotype Distribution:
Phenotypes:
3 wild-type (+/+ or +/m)
1 mutant (m/m)
Each cross yields a ratio of:
100% wild-type in specific combinations.
Common Pathways in Vertebrate Development
Similar development programs exist across vertebrates, suggesting that studying one system can provide insights into others.
Specific findings:
Mutations in zebrafish commonly display similar defects as seen in mutations in other vertebrate systems.
Techniques available for manipulation include:
Morpholinos: Effective in genetically modifying zebrafish.
Exposure to small molecules through water to investigate effects.
Zebrafish Cleavage
Cleavage pattern is telolecithal, whereby:
Most of the cell comprises yolk, and cleavage only occurs at the blastodisc—
The cytoplasm forms a free region at the animal pole.
Type of Cleavage:
Meroblastic
Discoidal: only the blastodisc forms the embryo.
Cleavage Stages
Synchronous Division: Cells divide synchronously until the 12th division.
Cells remain on the yolk cell, and by the 10th division,
Cell division slows as zygotic transcription begins, leading to:
Formation of the 1,000 cell stage and the mid-blastula transition.
There are three major layers forming at this stage:
Yolk Syncytial Layer (YSL): Contains nuclei that fuse with the yolk cell.
Enveloping Layer (EVL): A single layer which becomes the periderm.
Deep Cells: Give rise to the embryo proper.
Fate Map Definition
Fate mapping is established late in cleavage:
Early blastoderm cells remain undefined due to
Mixing of cells during cleavage.
Cell fates are fixed prior to gastrulation.
Zebrafish Gastrulation
The embryo shifts over the yolk through a process known as epiboly.
The YSL is attached to the EVL, which is pulled over the yolk.
The deep cells fill in as the YSL moves via microtubules.
One side of the embryo becomes thicker, designating the future dorsal side.
Germ Layer Formation
At 50% epiboly, a germ ring forms, which comprises the epiblast and hypoblast.
The cells begin to involute or ingress, causing:
Intercalation between epiblast and hypoblast to form a shield, which represents the future dorsal side of the organism and serves as a point of axis organization.
Cellular Migration
Cells around the margin migrate to form the hypoblast and extend anteriorly.
They narrow to express choradmesoderm and paraxial mesoderm.
The epiblast converges and extends, forming a neural keel; remaining cells become the ectoderm.
Involution and Tail Development
Endoderm involutes first, forming deep cells.
Epiboly continues and eventually closes off the bottom of the yolk, while
The head forms at the animal pole and the tail at the vegetal pole.
Dorsal-Ventral Axis Formation
The embryonic shield organizes the dorsal aspect of the embryo.
Transplantation of an additional shield can induce the formation of a secondary embryo.
Prechordal plate and notochord arise from the shield with surrounding tissue inducing these structures.
Factors Influencing D-V Axis
The formation of the D-V axis is influenced by
Bone Morphogenetic Proteins (BMPs) and Wnts, which contribute to ventralization.
BMP suppression leads to dorsalization;
BMP is inactivated by chordin secreted by chordamesoderm.
The relationship between BMP and chordin concentrations may play a role in neural tube patterning.
Shield Development in Zebrafish
In amphibians, the endoderm beneath the dorsal blastopore lip organizes the shield (known as the Nieuwkoop center).
Nuclear localization of β-catenin is critical in zebrafish for activating genes in the shield that inhibit BMP signaling.
Factors influencing this include a paracrine factor similar to nodal and homeodomain proteins.
Anterior-Posterior Patterning
Wnt signaling differs in anterior and posterior specification, where
Removal of Wnt through morpholinos results in the formation of incorrectly specified anterior or posterior structures.
Early Development in Avian
The chicken is a representative model organism because:
Development is accessible year-round at consistent temperatures, with
Large quantities available for surgical manipulation.
Avian Cleavage Characteristics
Fertilization occurs in the oviduct with the egg receiving albumin and shell coating.
The embryo undergoes telolecithal discoidal meroblastic cleavage resulting in:
A small blastodisc (2-3 mm) located at the animal pole.
Early cleavage results in a single-layer structure thickening to 5-6 layers.
Cavity Formation Under Blastoderm
A subgerminal cavity forms as water is absorbed from albumin, creating spacing under the blastodisc.
Succeeding cell death leads to the formation of a one-cell thick area pellucida, while the surrounding region forms the area opaca, retaining deep cells—marginal zone is significant for embryonic cell fate.
Hypoblast Formation
Cells from the blastoderm delaminate into the subgerminal space forming islands known as primary hypoblasts.
These are later displaced by secondary hypoblasts that migrate anteriorly from the posterior margin, creating a blastocoel between the epiblast and hypoblast.
Formation of Embryonic Structures
The embryo is derived from the epiblast, while
The hypoblast develops into extraembryonic tissues.
Avian Gastrulation
During gastrulation, avians, reptiles, and mammals develop a primitive streak:
Begins with thickening in the posterior marginal region where epiblast cells become globular and motile from convergent extension, advancing the streak forwards while halving its width.
Migration Through Primitive Groove
Within the streak, a primitive groove forms, providing an opening for cells to enter the blastocoel.
Anteriorly lies Hensen’s node, equivalent to the blastopore lip or embryonic shield; the streak directs major developmental axis:
Streak is anterior to dorsal
Streak defines dorsal—cells migrate to ventral
Streak forms medial structures.
Endoderm and Mesoderm Development
Endoderm and mesoderm ingress through the primitive groove, where:
Endoderm replaces the secondary hypoblast;
Mesoderm forms as a layer between the endoderm and epiblast.
Regression of Primitive Streak
Hensen’s node retreats from the center of the area pellucida to the posterior, laying down the notochord.
Endoderm and mesoderm continue to ingress; anterior regions begin significant differentiation earlier leading to organ formation;
The epiblast is entirely ectoderm by this stage.
Ectoderm Migration
Ectoderm migrates through epiboly, which entails the ectoderm proliferating and ultimately encircling the yolk.
This enclosure process takes almost 4 days and follows the pathways laid by fibronectin.
Removal of fibronectin disrupts ectodermal migration.
Dorsal-Ventral Axis Formation via pH
The pH environment influences D-V axis formation:
The albumen above the epiblast has a basic pH of 9.5, while the subgerminal cavity is at an acidic pH of 6.5.
Water and Na⁺ are transported into the cavity, generating a 25 mV potential difference (positive on the ventral side).
Gravity Impact on Axis Formation
Gravity plays a role in structuring the anterior-posterior axis:
Initially, the blastoderm is radially symmetrical but is rotated in the shell gland for 20 hours at 10-12 revolutions per hour.
This leads to lighter yolk components settling at one end, initiating posterior end formation with the posterior marginal zone (PMZ) emerging.
Formation of the Node
The node in avians resembles the zebrafish shield and the amphibian dorsal blastopore lip.
The posterior region is induced to form the Nieuwkoop center, characterized by nuclear localization of β-catenin.
Misexpression of Vg1 results in the induction of a secondary node and primitive streak.
Dorsal-Ventral Axis Organization
The node organizes the dorsal-ventral axis:
Produces antagonists of BMP signaling such as noggin and chordin.
Inhibition of BMP allows the development of dorsal phenotypes while BMP signaling facilitates ventralization, with additional involvement by nodal to pattern the mesoderm.
Left-Right Axis Formation
Determined easily in chicks for experimental manipulation, with major influences from:
Transcription factors Pitx2 and Nodal (a paracrine factor from the TGF-b family).
Sonic hedgehog signaling halts on the embryo's right side due to activin and receptor expression.
This activates fgf8 signaling, with downstream regulation by snail.
On the left side, Lefty-1 inhibits fgf8 expression while nodal and Lefty-2 activate Pitx2.
Mammalian Development - Cleavage
Humans exhibit some of the smallest embryos (approximately 100 µm).
Fewer embryos are produced, and most develop internally.
Cleavage patterns vary from typical ones observed in other species, starting with:
Meridional cleavage for the first and rotational cleavage for the second.
Characteristics of Early Cleavage
Early cleavage is often asynchronous, presenting potential odd numbers of blastomeres.
Transition from maternal to genomic control noted at the 2-cell stage without a mid-blastula transition.
At the 8th cell division, E-cadherin expression initiates compaction.
Morula Formation
At the 16-cell stage, the structure becomes a morula:
Inner cells are surrounded by outer cells.
The outer layer forms the trophoblast, which is the embryonic portion of the placenta, while the inner cells comprise the inner cell mass (ICM).
This stage marks the first differentiation event; the ICM remains pluripotent.
Blastocyst Development
The morula, initially without an internal cavity, evolves as the trophoblast layer secretes fluid to create a blastocoel:
Na/K ATPase pumps Na⁺ into the central cavity, attracting water and forming the blastocyst, with the inner cell mass positioned to one side.
Embryo Implantation
Implantation in the endometrium is initially hindered by the zona pellucida:
The blastocyst secretes proteases to puncture the zona and facilitate escape.
It binds to the endometrium ECM through integrins in the trophoblast, which also secretes additional proteases to aid in embedding within the endometrium.
Mammalian Gastrulation
Mammal gastrulation resembles reptiles, given that mammals originate from reptilian lineage;
The yolk sac is empty, with the embryo relying on maternal nutrients, resulting in uterine formation and the development of new fetal organs.
The inner cell mass separates into an upper layer (epiblast) forming the embryo and the amnionic cavity filled with amnionic fluid and a lower layer (hypoblast) responsible for forming the yolk sac.
Node Formation in Mammals
The node develops at the posterior while cells migrate through the primitive groove, with early cells displacing the hypoblast to form the endoderm, while later cells form the mesoderm between the endoderm and epiblast.
Extraembryonic Membranes in Mammals
The trophoblast differentiates into:
Cytotrophoblasts
Syncytiotrophoblasts: Multinucleated cells that invade the uterine wall and remodel blood vessels.
Maternal blood bathes fetal blood vessels, with extraembryonic mesoderm derived from the yolk sac and primitive streak cells contributing to the formation of the umbilical cord.
Anterior-Posterior Signaling in Mouse
The most extensively studied signaling pathway in early mammalian development.
Centers of signaling include anterior visceral endoderm (AVE) and the node.
AVE forms before the streak and induces nodal in the epiblast, which patterns visceral endoderm.
Shifts in visceral endoderm expression will alter epiblast expression accordingly.
Node Similarity in Developmental Biology
The node functions similarly to the shield or blastopore lip found in other vertebrates, producing chordin and noggin.
The AVE expresses genes critical for head formation; both the node and AVE arise on opposite sides of the embryos.
Hox genes contribute further to defining anterior-posterior polarity across vertebrate species.
Dorsal-Ventral Axis Formation Knowledge
Less is known about how the dorsal-ventral axis forms compared to the anterior-posterior axis:
The axis derives from the positioning of the inner cell mass.
Major regions include the animal pole, vegetal pole, and presumptive dorsal and ventral regions.
Left-Right Axis Formation
Determined in chicks with findings indicating that the initiation is influenced by:
Ciliary movements on nodal cells activating specific pathways on either side of the embryo.