Developmental Biology Lecture #7
Early Development of Fish, Birds, and Mammals
Each undergoes meroblastic cleavage.
A small portion of the embryo divides.
Most of the yolk remains undivided.
Early Development in Fish
Teleost Danio rerio (Zebrafish): A convenient model system due to:
Breeds year-round.
Exhibits external development.
Produces transparent embryos.
Is easy to maintain.
Develops rapidly: free-swimming larvae by 5 days, with functional organs.
Large Scale Mutagenesis Screen
Zebrafish as the first vertebrate screened for developmental mutants.
Parent males exposed to a chemical mutagen.
Generational crosses:
Parents: Mutagenized (+/m) and Wild-type (+/+).
F1 Generation: Mutagenized (+/m) and Wild-type (+/+).
F2 Generation: Possible combinations include +/m, +/m, +/+, m/m.
F3 Generation: Phenotypic ratios:
3 wild-type (+/+ or +/m) to 1 mutant (m/m).
Common Pathways in Vertebrate Development
Similar developmental programs across vertebrates.
Studying one system yields information about others.
Mutations in zebrafish can reflect defects observed in other systems.
Zebrafish are manipulable:
Morpholinos (antibody molecules) work well.
Exposure to small molecules in water enables targeted research.
Zebrafish Cleavage
Telolecithal cleavage: most cells consist of yolk.
Cleavage is restricted to the blastodisc (cytoplasm-free region at animal pole).
Type of cleavage: meroblastic and discoidal (only blastodisc forms the embryo).
Synchronization of Divisions
Cells divide synchronously until the 12th division.
Cells remain on the yolk cell, and by the 10th division, cell division slows down, initiating zygotic transcription.
At the 1000-cell stage: midblastula transition.
Formation of:
Yolk syncytial layer (YSL): nuclei fuse with the yolk cell.
Enveloping layer (EVL): a single layer that becomes the periderm.
Deep cells: contribute to the embryo proper.
Fate Mapping
Fate maps defined late in cleavage.
Early blastoderm cells remain undifferentiated; there is mixing of cells during cleavage.
Cell fates become fixed before gastrulation begins.
Gastrulation in Zebrafish
The embryo moves over the yolk by epiboly.
The YSL, attached to EVL, is pulled over the yolk.
Deep cells fill in the space below.
Movement facilitated by microtubules; the future dorsal side thickens.
Germ Layer Formation
By 50% epiboly, a germ ring forms from epiblast and hypoblast.
Cells begin involuting or ingressing:
Epiblast and hypoblast intercalate to form a shield at the future dorsal side, which acts as an axis organization point.
Cellular Movement and Layer Formation
Cells around the margin move to the hypoblast:
Migrate to the shield and extend anteriorly.
Form chordamesoderm (anterior) and paraxial mesoderm (sides).
Additional movement results in:
Neural cells forming the neural keel.
Remaining cells creating the ectoderm.
Endoderm and Ectoderm Migration
The endoderm involutes first, forming deep cells.
Epiboly continues, closing off the bottom of the yolk.
The head forms at the animal pole; the tail develops at the vegetal pole.
Dorsal-Ventral Axis Formation
The embryonic shield organizes the dorsal side; transplanting another shield creates a second embryo.
Formation of prechordal plate and notochord from the shield influences surrounding tissue induction.
Mechanisms of Axis Patterning
Patterning of D-V axis primarily influenced by:
BMPs and Wnts: ventralize the embryo.
BMP inhibition results in dorsalization, where chordin, produced by chordamesoderm, inactivates BMP.
The ratio of BMP to chordin is crucial for neural tube patterning.
Establishing the Shield
In amphibians, the endoderm beneath the dorsal blastopore lip organizes the shield (Nieuwkoop center).
Nuclear localization of β-catenin is crucial for gene activation that inhibits BMPs, with induction factors like nodal being also involved.
Anterior-Posterior Patterning in Zebrafish
Different Wnts are used for anterior vs. posterior specification.
Removal of Wnts using morpholinos leads to a loss of distinct anterior or posterior structures.
Early Development in Avian Species (Chick)
Chick serves as a representative model:
Accessible year-round; development governed by temperature, ensuring stages are uniform.
Large numbers of embryos can be surgically manipulated.
Cleavage in Avian Species
Fertilization occurs in the oviduct; secretion includes albumin and shell around the egg.
Cleavage type: telolecithal, discoidal, meroblastic.
A small blastodisc (2-3 mm) forms at the animal pole and undergoes an early cleavage that creates a single layer thick, expanding to 5 to 6 cell layers thick.
Formation of Subgerminal Cavity
Under the blastoderm, a subgerminal cavity forms:
Cells absorb water from the albumen and secrete fluid, resulting in a zone that becomes the area pellucida (one cell thick) and an outer ring called area opaca.
The marginal zone contains cells at the border of these two areas, which are significant for embryonic cell fate.
Hypoblast Formation
Cells from blastoderm delaminate to the subgerminal space, forming isolated islands of primary hypoblast,
Displaced by cells from the posterior margin, forming the secondary hypoblast that moves anteriorly, creating the blastocoel between the epiblast and hypoblast.
Roles of the Hypoblast and Epiblast
The epiblast forms the embryo, while the hypoblast develops into extraembryonic tissues.
Gastrulation in Avian Species
During gastrulation, avians, reptiles, and mammals develop a primitive streak:
Initiated by thickening in the posterior marginal region of the epiblast.
Cells become globular and motile through convergent extension as the streak forms.
The primitive groove forms, where cells migrate to the blastocoel, and the anterior end is known as Hensen’s node (equivalent to blastopore lip).
Movement and Migration Patterns
Endoderm and mesoderm pass through the primitive groove:
Endoderm displaces secondary hypoblast cells.
Mesoderm forms a loose layer between the endoderm and epiblast.
Regression of the Primitive Streak
The primitive streak regresses while Hensen’s node moves towards the posterior, laying down the notochord.
Endoderm and mesoderm continue ingressing, with anterior regions differentiating earlier into organs.
The epiblast ultimately contributes entirely to the ectoderm.
Ectodermal Migration
The ectoderm proliferates via epiboly, encapsulating the yolk within approximately 4 days, guided by fibronectin tracks.
Removal of fibronectin prevents ectodermal migration.
Dorsal-Ventral Axis Formation in Avian Development
pH levels influence the D-V axis formation:
Albumen above the epiblast has a basic pH of 9.5, while the subgerminal cavity has an acidic pH of 6.5.
Potentials establish with ion transport creates a 25 mV difference, positive on the ventral side.
Gravity and Axis Orientation
Gravity impacts the anterior-posterior axis formation:
The blastoderm starts radially symmetric but rotates in the shell gland, causing lighter yolk components to migrate to one end, forming the posterior marginal zone (PMZ).
Node Formation
The node is similar to the zebrafish shield and the amphibian dorsal blastopore lip:
Induction of the posterior region leads to the formation of the Nieuwkoop center driven by nuclear localization of β-catenin.
Co-expression of posterior Vg1 with β-catenin induces the node formation, while misexpression leads to secondary node formation.
Organization of the Dorsal-Ventral Axis by the Node
The node produces antagonists of BMP signaling, specifically noggin and chordin.
BMP inhibition facilitates dorsal phenotypes, while BMP signaling allows ventralization. Nodal factors help in mesoderm patterning.
Left-Right Axis Formation in Avian Development
Access to manipulation makes it easier to determine the left-right axis in birds:
Governed by transcription factors Pitx2 and Nodal (as paracrine factors from the TGF-β family).
Sonic hedgehog expression ceases on the right side, activated downstream of FGF8, while Lefty-1 inhibits FGF8 on the left, maintaining nodal expression.
Mammalian Development: Cleavage
Mammalian embryos are among the smallest, with human embryos measuring only 100 mm and developing internally.
Cleavage differs from other species:
Fertilization occurs in the ampulla of the oviduct, close to the ovary.
Divisions occur relatively slowly, with intervals of 12-24 hours and varying patterns (meridional firstly, then rotational).
Cleavage Synchronization and Cell Count
Early cleavage is asynchronous; embryos often have odd numbers of blastomeres due to the switch from maternal to genomic control at the 2-cell stage.
No mid-blastula transition is present; E-cadherin expression begins by the 8th cell division, leading to compaction.
Morula to Blastocyst Formation
At 16 cell stage (morula), inner cells become surrounded by outer trophoblast cells:
Outer cells form the trophoblast (the embryonic portion of the placenta), while inner cells (inner cell mass - ICM) form the embryo itself and remain pluripotent.
The morula initially lacks an internal cavity.
The trophoblast layer secretes fluid to develop into a blastocoel, facilitated by Na/K ATPase pumping sodium into the cavity, leading to osmotic water influx.
This process repositions the ICM to one side, forming a blastocyst.
Implantation Process in Mammals
The blastocyst is hindered from implantation by the zona pellucida, which it escapes by secreting protease to form an exit.
The blastocyst binds to the endometrium extracellular matrix via integrins in the trophoblast, with additional proteases facilitating embedding in the endometrium.
Gastrulation in Mammals
Mammal gastrulation mirrors that of reptiles:
Absence of a yolk sac; embryos derive nutrients maternally, promoting uterus formation and establishing the fetal organ.
The inner cell mass divides into two layers:
Hypoblast (lower layer forming the yolk sac) and epiblast (upper layer forming the embryo and amniotic cavity).
Node and Migration During Gastrulation
The node emerges in the posterior as cells migrate through the primitive groove:
Early moving cells replace hypoblast to form the endoderm, while later cells establish a mesoderm layer in between the endoderm and epiblast.
Extraembryonic Membranes in Mammals
Unique structures arise from trophoblasts:
Cytotrophoblasts and syncytiotrophoblasts.
Syncytiotrophoblasts are multinucleated cells invading the uterine wall, remodeling blood vessels for nutrient exchange.
Extraembryonic mesoderm forms from yolk sac and primitive streak cells, fusing with trophoblast extensions to form the umbilical cord.
Key Structures in Placental Exchange
Chorionic villi: facilitate maternal-fetal exchange.
Maternal blood flows through maternal arteries and veins to the fetal circulation via umbilical arteries and veins through the intervillus space.
Anterior-Posterior Signaling in Mouse Development
Most known from studies in mice:
The proximal inner cell mass and adjacent structures change dynamically during the 3.5 to 6.5 days period, facilitating key developmental landmarks.
Key Signaling Centers
The anterior visceral endoderm (AVE) precedes the development of the streak and node:
Extraembryonic ectoderm triggers nodal expression in the epiblast, creating effects that pattern visceral endoderm and cause shifts in epiblast expression.
Node Functionality in Development
The node exhibits similarities to the shield, blastopore lip, and chicken node:
Produces chordin and noggin.
The AVE expresses genes necessary for head formation, with both the node and AVE located on opposite sides of the embryo body.
Hox genes further offer specificity in anterior-posterior patterns across vertebrates.
Dorsal-Ventral Axis Establishment
Knowledge of the dorsal-ventral axis remains limited:
It is derived from the inner cell mass position and cell distribution during early cleavage.
Left-Right Axis Specification
The left-right axis is regulated by similar gene factors observed in avian studies:
Initiated by cilia movements in nodal cells that activate asymmetric signaling pathways, thus determining left-right axis differentiation in the embryo.