Lecture 7: Early Development in Fish, Birds, and Mammals

Early Development in Fish, Birds, and Mammals

  • Meroblastic cleavage:
      - A type of cleavage where only a small portion of the embryo divides, while most of the yolk remains undivided.

Early Development in Fish

  • Teleost Danio rerio (Zebrafish):
      - A convenient system for developmental biology because:
        1. They breed all year round.
        2. They exhibit external development.
        3. The embryos are transparent.
        4. They are easy to maintain.
        5. They have fast development; from 1 cell to free swimming larvae with functional organs in just 5 days.

Large Scale Mutagenesis Screen

  • First vertebrate analyzed for developmental mutants.

  • Process:
      - Males were exposed to a chemical mutagen.
      - Parental generation includes:
        - Mutagenized and wild-type where wild-type is represented as +/+ and mutant alleles as +/m.   - F3 generation breeding options include:
        - +/m x +/+
        - +/+ x +/+
        - +/+ x +/m
        - +/m x +/m

  • Mutagen effects are observed in subsequent generations.

Common Pathways in Vertebrate Development

  • Vertebrates share similar developmental programs.

  • By studying one system (like the zebrafish), insights can be gained about others.

  • Mutations in zebrafish often result in similar defects in other vertebrate systems.

  • Manipulative techniques include:
      1. Effective use of morpholinos.
      2. Exposure to small molecules in water.

Zebrafish Cleavage

  • Cleavage Type:
      - Teleolecithal (most of the cell is yolk).
      - Cleavage occurs only in the blastodisc, the cytoplasm region free of yolk.
      - This type of cleavage is meroblastic and discoidal, leading to embryo formation from only the blastodisc.

  • Cleavage process:
      - Cells divide synchronously until the 12th division, remaining on the yolk cell.
      - Division slows at the 10th division, initiating zygotic transcription leading to the 1000 cell stage (midblastula transition).

  • Key structures formed:
      1. Yolk Syncytial Layer (YSL): Formed by nuclei fusing with cells of the yolk cell.
      2. Enveloping Layer (EVL): A single layer that becomes the periderm.
      3. Deep Cells: These give rise to the embryo proper.

Fate Mapping in Zebrafish

  • Fate map is defined late in cleavage.

  • Early blastoderm cell fates are undetermined due to cell mixing.

  • Cell fates become fixed prior to gastrulation.

Zebrafish Gastrulation

  • The embryo migrates over the yolk via a process known as epiboly.

  • YSL is connected to EVL and pulled over the yolk.

  • Deep cells fill in as YSL moves, driven by microtubule action.

  • One side of the embryo thickens, indicating the future dorsal side.

Germ Layer Formation

  • As embryo undergoes 50% epiboly, a germ ring forms, comprising the epiblast and hypoblast.

  • Cells begin to involute or ingress, resulting in intercalation and the formation of the shield, setting up the future dorsal side and axis organization.

  • Cells around the margin migrate towards the hypoblast, eventually forming the choradmesoderm and paraxial mesoderm, with the epiblast converging and extending to form the neural keel. Remaining cells differentiate to give rise to the ectoderm.

Formation of Endoderm and Mesoderm

  • Endoderm involutes first, forming deep cells.

  • Epiboly proceeds to close off the bottom of the yolk, with the head developing at the animal pole and the tail at the vegetal pole.

Dorsal-Ventral Axis Formation

  • The embryonic shield organizes the dorsal side.

  • Transplantation of a shield can create a second embryo.

  • Structures formed from the shield include the prechordal plate and notochord, which induce surrounding tissues.

Patterning the Dorsal-Ventral Axis

  • Bone Morphogenetic Proteins (BMPs) and Wnts contribute to ventralization of the embryo.

  • Suppressing these factors allows for dorsalization, where BMP inhibition occurs through chordin secreted by chordamesoderm.

  • The ratio of BMP to chordin is significant in patterning the neural tube.

Establishment of the Shield

  • In amphibians, the endoderm beneath the dorsal blastopore lip organizes the shield known as the Nieuwkoop center.

  • The nuclear localization of β-catenin is found in the nuclei of zebrafish YSL, activating shield-specific genes that inhibit BMPs alongside nodal-like paracrine factors and homeodomain proteins.

Anterior-Posterior Patterning

  • Different Wnt proteins are utilized for the specification of anterior and posterior structures.

  • The removal of certain signals with morpholinos results in the development of improperly defined anterior or posterior structures.

Early Development in Avian Species

  • Chickens serve as a model organism due to:
      - Year-round accessibility.
      - Consistent developmental stages at specific temperatures.
      - Surgical manipulability.

Fertilization and Early Cleavage

  • Fertilization occurs in the oviduct, where albumen and shell are secreted around the egg.

  • The cleavage is telolecithal discoidal meroblastic, leading to the formation of a blastodisc.

  • Initial cleavage results in a single layer thick, eventually thickening to 5 to 6 cell layers.

Subgerminal Cavity Formation

  • A subgerminal cavity is formed under the blastoderm as cells absorb water and secrete fluid, leading to a one cell-thick area called the pellucida.

  • An outer ring known as the area opaca retains deep cells, with a marginal zone at the border crucial for embryonic cell fate.

Hypoblast Formation

  • Cells from the blastoderm delaminate into the subgerminal space to form isolated islands called primary hypoblast.

  • These are displaced by cells from the posterior margin forming a secondary hypoblast, migrating anteriorly and creating the blastocoel between the epiblast and hypoblast.

Gastrulation in Avian Species

  • Gastrulation in birds, reptiles, and mammals initiates with the formation of a primitive streak at the posterior marginal region.

  • The epiblast thickens and cells become motile, resulting in convergent extension that advances the streak forward, thereby narrowing its width.

  • Inside the streak forms a primitive groove, allowing cells to pass into the blastocoel.

  • The anterior end features Hensen’s node, equivalent to the blastopore lip or embryonic shield marking anterior and dorsal structures.

Movement and Formation of Endoderm and Mesoderm

  • Cells move through the primitive groove, where endoderm displaces secondary hypoblast cells, and mesoderm forms as a separate layer between the endoderm and epiblast.

  • The primitive streak regresses, and Hensen's node moves from the center of the area pellucida to the posterior as the notochord is established.

Ectoderm Formation and Development

  • The ectoderm undergoes epiboly, proliferating and surrounding the yolk, completing this process in nearly four days while following fibronectin tracks for attachment.

  • Disruption in fibronectin contacts inhibits ectodermal migration.

Dorsal-Ventral Axis Formation

  • The pH gradient influences D-V axis formation.
      - Basic pH (9.5) in the albumen above the epiblast.
      - Acidic subgerminal cavity (pH 6.5) wherein a 25 mV potential difference exists, whereby the ventral side is positive.

Gravity and Anterior-Posterior Axis Formation

  • Initially radially symmetric, the blastoderm undergoes rotation in the shell gland, influencing the distribution of yolk components.

  • The lighter yolk components settle to one end, forming the posterior marginal zone (PMZ).

Node Formation

  • The node shares similarities with the zebrafish shield and amphibian dorsal blastopore lip, where posterior regions induce the formation of the Nieuwkoop center.

  • Nuclear localization of β-catenin with posterior Vg1 expression initiates the node's formation; misexpression of Vg1 can trigger secondary node formation.

Node Functionality and Axis Organization

  • The node organizes the dorsal-ventral axis by producing antagonists of BMP signaling like noggin and chordin, allowing for dorsal phenotypes and patterning of the mesoderm with nodal expression.

Left-Right Axis Formation

  • Easily studied in the chick model; determined by transcription factors such as Pitx2 and Nodal from the TGF-b family.

  • Asymmetric expression of sonic hedgehog (Shh) on the right and activin on the left, affecting downstream gene pathways and establishing left-side specific factors like Lefty-1.

Mammalian Development - Cleavage

  • Mammalian embryos are notably small (e.g., humans measuring 100 µm) and develop internally.

  • Cleavage follows different patterns compared to other classes; fertilization occurs in the ampulla of the oviduct, with divisions happening slowly (averaging 12-24 hours apart).

Cleavage Patterns

  • Initial cleavage divisions are meridional followed by rotational orientations.

  • In early stages, cleavage is asynchronous with odd numbers of blastomeres being common.

  • A switch from maternal to genomic control occurs at the 2-cell stage without a mid-blastula transition.

Morula Formation

  • At the 8th cell division, E-cadherin expression commences leading to the compaction of the embryo.

  • By the 16-cell stage, termed the morula, inner cells are surrounded by outer cells, which differentiate into trophoblast and inner cell mass (ICM), where the ICM remains pluripotent.

Blastocyst Formation and Implantation

  • Morula proceeds to no internal cavity formation, but the trophoblast layer secretes fluid to form a blastocoel through Na/K ATPase activity that draws water inside, positioning the ICM to one side, resulting in the blastocyst.

  • The blastocyst cannot implant due to the zona pellucida, escaping by protease secretion which allows it to bind to the endometrium ECM with integrins. Further proteases assist in embedding the blastocyst into the endometrium.

Mammalian Gastrulation

  • Sharing traits with reptiles, mammalian gastrulation involves low yolk sac presence, receiving nutrients maternally.

  • Results in the formation of the uterus and additional fetal organs.

  • The ICM differentiates into two layers: the hypoblast (forming the yolk sac) and epiblast (forming the main embryo and amniotic cavity).

Node and Cell Migration

  • The node forms in the posterior with cells migrating through the primitive groove, displacing hypoblast contributions to establish endoderm and mesoderm.

Unique Extraembryonic Membranes in Mammals

  • Trophoblast cells subdivide into cytotrophoblasts and multinucleated syncytiotrophoblasts, embedding into the uterine wall, remodeling maternal blood vessels for nutrient exchange.

  • Extraembryonic mesoderm is formed from yolk sac and primitive streak cells, contributing to the umbilical cord's formation.

Placental Anatomy

  • Chorionic Villus:
      - Acts as a site for nutrient and gas exchange between maternal and fetal blood, structured with fetal blood vessels bathed in maternal blood.

  • Vascular Structure:
       - Umbilical arteries and vein structure facilitates efficient delivery and exchange of blood between mother and fetus, with maternal arteries providing blood to chorionic villi for perfusion.