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Amphibians & Fish Early Development and Axis Formation
Chapter 7 – Part 1
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Amphibian and Fish Backbone of vertebrate embryology Frogs (Xenopus)
Large cells and rapid development But, long period before fertile Zebra fish (Danio rerio) Model organism for study of vertebrate development Breed each year Easily maintained Transparent embryos At 24 hours after fertilization – embryo has formed most of its organ primordia
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Fertilization, Cortical Rotation, and Cleavage
Fertilization and cortical rotation Can occur anywhere in animal hemisphere of amphibian Sperm entry point determines orientation of the dorsal ventral axis Marks ventral side 180° opposite sperm entry point marks the dorsal side Centrioles organize microtubules in egg Arrange in parallel array in vegetal cytoplasm Cortical cytoplasm Rotates 30° relative to internal cytoplasm Array disappears when rotation stops
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Fertilization, Cortical Rotation, and Cleavage
7.1 Formation of the parallel arrays of microtubules in the vegetal hemisphere along the future dorsal-ventral axis 40% first cell cycle complete – microtubules begin forming 50% first cell cycle complete – more microtubules, but lack polarity 70% first cell cycle complete – parallel array of microtubules
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Fertilization, Cortical Rotation, and Cleavage
Opposite sperm entry site Gray crescent Where gastrulation will begin 7.1 Reorganization of cytoplasm in the newly fertilized frog egg
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Fertilization, Cortical Rotation, and Cleavage
7.1 Reorganization of cytoplasm in the newly fertilized frog egg
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Unequal Radial Holoblastic Cleavage
Radially symmetrical and holoblastic Contains more yolk Concentrated in vegetal pole 1st cleavage bisects gray crescent 2nd cleavage starts before 1st finishes Uneven division due to presence of yolk in vegetal hemisphere Even with unequal size Blastomeres continue to divide at same rate
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Unequal Radial Holoblastic Cleavage
7.2 Cleavage of a frog egg
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Unequal Radial Holoblastic Cleavage
7.3 Scanning electron micrographs of frog egg cleavage First cleavage Second cleavage (4cells) Fourth cleavage (16 cells)
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Unequal Radial Holoblastic Cleavage
Unequal radial holoblastic cleavage (cont’) Small micromeres at animal pole Large macromeres at vegetal pole Regulated by MPF (mitosis promoting factors) At cell stage called MORULA (mulberry)
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Unequal Radial Holoblastic Cleavage
At 128 cell stage Blastocoel formed Embryo considered a Blastula 2 functions: Permit cell migration during gastrulation Prevent cells beneath it from interacting prematurely with cell above Roof of blastocoel in animal hemisphere Animal cap If placed near bottom of vegetal cells – cap cells become mesoderm Cleavage to Blastula
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Unequal Radial Holoblastic Cleavage
Mesoderm formed next to vegetal endoderm precursors Animal cap destined to become nerves and skin Cell adhesion molecules keep cells together EP cadherin 7.4 Depletion of EP-cadherin mRNA in the Xenopus oocyte results in the loss of adhesion between blastomeres and the obliteration of the blastocoel
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Mid Blastula Transition: Preparing for Gastrulation
Very few genes transcribed during early cleavage Nuclear genes are not activated until the 12th cell cycle Acquires capacity to become motile Mid blastula transition (MBT) Egg factors initiate this (absorbed by newly made chromatin) Different genes activated in different cells Cell cycle acquires gap phases Blastomeres acquire capacity to become motile
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Amphibian Gastrulation
Multiple ways of gastrulation in different amphibians Xenopus fate map Goal is to bring inside embryo areas destined to form endodermal organs Blastula has different fates depending on whether a cell is located deep or superficially in the layers Mesoderm precursors exist mainly in deep layers Endoderm (bottom) and Ectoderm (top)
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Vegetal Rotation and the Invagination of the Bottle Cells
Gastrulation movements in the frog To position the mesoderm between the outer ectoderm and the inner endoderm Vegetal rotation and the invagination of the bottle cells Gastrulation starts on the future dorsal side of embryo Below equator Region of gray crescent Region opposite the sperm entry point
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Amphibian Gastrulation
7.6 Cell movements during frog gastrulation
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Vegetal Rotation and the Invagination of the Bottle Cells
At this point, cells invaginate to form a slitlike blastopore called bottle cells Change shape Maintain contact with outside cells Bottle cell’s line the archenteron Similar to sea urchins except gastrulation in frogs begins in marginal zone not ventral region Endoderm not as large/yolky here as other vegetal blastomeres
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Vegetal Rotation and the Invagination of the Bottle Cells
Experiments (Hardin & Keller) proved: Dorsal lip cell removed placed on inner endoderm Formed bottle cells and sank below the surface Once involution starts Gastrulation will proceed without bottle cells Coordinated involution of subsurface cells more important
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Vegetal Rotation and the Invagination of the Bottle Cells
Initiated by internalization of endoderm and mesoderm 2 hours before bottle cells are formed Rotation puts prospective pharyngeal endoderm adjacent to the blastocoel and above involuting mesoderm Marginal cells form endodermal lining of archenteron If the involuting marginal zone (IMZ) cells were removed Archenteron formation would stop
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Vegetal Rotation and the Invagination of the Bottle Cells
7.8 Early movements of Xenopus gastrulation
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Involution at the Blastopore Lip
Involution of blastopore lip Involution of marginal zone cells Animal cells undergo Epiboly and converge at blastopore Dorsal lip of blastopore Migrating Margin cells turn inward and travel along inside animal hemisphere Form the lip of the blastopore Constantly changing
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Amphibian Gastrulation
7.6 Cell movements during frog gastrulation
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Amphibian Gastrulation
7.6 Cell movements during frog gastrulation
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Amphibian Gastrulation
7.6 Cell movements during frog gastrulation 7.7 Surface view of an early dorsal blastopore lip of Xenopus
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Involution at the Blastopore Lip
Dorsal lip of blastopore Margin cells turn inward and travel inside animal hemisphere Form the lip Will become cells that involute to form prechordal plate Precursor of the head mesoderm Followed by next cells to involute in at lip to become chordamesoderm Will form the notochord Important to inducing and patterning nervous system
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Amphibian Gastrulation
7.8 Early movements of Xenopus gastrulation
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Involution at the Blastopore Lip
Slowly blastocoel is displaced to opposite side of the dorsal lip Lateral lips and ventral lips form on “crescent” to make additional mesoderm and endodermal cells Finally a ventral lip covers over additional mesodermal and endodermal precursor cells Forming a “ring” blastopore Remaining endoderm patch is called the yolk plug Eventually internalizes
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Amphibian Gastrulation
7.9 Epiboly of the ectoderm
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Convergent Extension of the Dorsal Mesoderm
Involution begins dorsally Pharyngeal endoderm and head mesoderm Next tissue to enter forms the notochord and somite Lip of the blastopore expands to have dorsolateraly, lateral, and ventral sides Prospective heart, kidney, and ventral mesoderm that enters the embryo
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Convergent Extension of the Dorsal Mesoderm
Blastopore lip IMZ cells (involuting marginal zone) Several layers deep IMZ cells intercalate radially Continues to extend vegetally As these cells reach blastopore lip – involute inwardly initiation 2nd intercalation Causes convergent extension along mediolateral axis Simultaneous elongation with involution Mesoderm continues to migrate to animal pole Forms an endodermal roof of the archenteron
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Convergent Extension of the Dorsal Mesoderm
7.10 Xenopus gastrulation continues
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Convergent Extension of the Dorsal Mesoderm
Central Dorsal mesoderm Become notochord and somites Remainder of body mesoderm Forms heart, kidneys, bones, ….. Entered through ventral and lateral blastopore lips These create the mesodermal mantle Endoderm formed from involuting marginal zone (IMZ) that forms lining of archenteron roof Endoderm from vegetal cells that become archenteron floor Where endoderm meets ectoderm becomes anus
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Epiboly of the prospective ectoderm
The animal cal and noninvoluting marginal zone (IMZ) cells expand by epiboly Covers the entire embryo Form the surface ectoderm By increasing the cell number (through division) coupled with concurrent integration of several deep layers into one Involves assembly of fibronectin into fibrils
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Epiboly of the prospective ectoderm
7.13 Epiboly of the ectoderm is accomplished by cell division and intercalation
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Progressive Determination of the Amphibian Axes
Remember This process whereby the central nervous system forms through interactions with the underlying mesoderm Called Primary Embryonic Induction A principle way vertebrate body becomes organized Discoverers called the dorsal blastopore lip “The Organizer”
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Hans Spemann and Hilde Mangold: Primary Embryonic Induction
Began his most famous work in 1903 on demonstrating nuclear equivalence Later he won the Nobel Prize in 1935 Later his work (1924) centered on the dorsal lip cells, referred to as the Spemann’s Organizer Hilde Mangold PhD Student of Spemann Began assisting him in his work in 1924 and contributed to understanding cell fate in early gastrulation These cells originated as self-differentiating tissue of the dorsal lip Derived from the gray crescent cytoplasm
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Hans Spemann and Hilde Mangold: Primary Embryonic Induction
Spemann Organizer Role of the dorsal lip cells destined to be: Dorsal mesoderm Notochord Some anterior pharyngeal Induce host’s ventral tissue to change fate and form neural tube and dorsal mesoderm (such as somites) Organize host and donor tissues into 2nd embryo with AP and DV axes using donor tissue
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Hans Spemann and Hilde Mangold: Primary Embryonic Induction
7.17 Organization of a secondary axis by dorsal blastopore lip tissue
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Hans Spemann and Hilde Mangold: Primary Embryonic Induction
Spemann Organizer (cont) Responsible for neural tube formation Chordamesoderm & ectoderm cannot organize the entire embryo Responsible for transforming flanking mesoderm into AP axes These events initiate a sequential series of sequential inductive events This is Key Inductive Event Dorsal lip cells inducing the A-P axis and neural tube Called Primary Embryonic Induction
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Molecular Mechanisms of Amphibians Axis Formation
This actually created more questions than answers by these experiments How does the organizer form? ~ dozen cells of the initial organizer position themselves opposite the point of sperm entry Now discovered that they are in the right position for 2 signals converging First signal – tells the cells that they are dorsal Second signal – tells them they are mesoderm
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The Dorsal Signal: -Catenin
Two signals & others: Dorsal signal: -caterin Isolating cap, marginal, vegetal cells Organizer cells are special because they reside above a special group of vegetal cells Recombined animal cap with vegetal cells Vegetal cells induced mesoderm formation from animal cap cells (marginal/equatorial cells) Ventral vegetal – blood, mesenchyme Intermediate vegetal – muscle, kidney Dorsal vegetal – somites, notochord These dorsal inducing vegetal cells called the Nieuwkoop center
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The Dorsal Signal: -Catenin
7.18 Summary of experiments by Nieuwkoop and by Nakamura and Takasaki, showing mesodermal induction by vegetal endoderm
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The Dorsal Signal: -Catenin
β-caterin Activin-like TGF-β &FGFs 7.18 Summary of experiments by Nieuwkoop and by Nakamura and Takasaki, showing mesodermal induction by vegetal endoderm
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The Dorsal Signal: -Catenin
Proof of dorsal vegetal cells is inducer of organization Nieuwkoop center Similar proof of dorsal-most vegetal cells induce animal cell to form dorsal mesoderm -caterin Protein acting as cell anchor for cadherins Found to give this property to dorsal vegetal cells [In sea urchin responsible for specifying micromeres]
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The Dorsal Signal: -Catenin
If -catenin is originally found throughout the embryo, how does it become localized specifically to the side opposite the sperm entry? Answer translocation of Wnt11 and the Disheveled (Dsh) protein from the vegetal pole to the dorsal side of the egg at fertilization -catenin is targeted for destruction by glycogen synthase kinase 3 (GSK3) GSK3 destroys b-catenin and blocks axis formation GSK3 is inactivated by GSK3-binding protein (GBP) Occurs during the 1st cell cycle when microtubules are formed
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The Dorsal Signal: -Catenin
7.21 Model of the mechanism by which the Disheveled protein stabilizes -catenin in the dorsal portion of the amphibian egg
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The Dorsal Signal: -Catenin
GSK3-binding protein (GBP) Travels along the microtubules by binding to kinesin Moves to a point opposite sperm entry Disheveled grabs the GPB and is translocated on microtubules Cortical rotation is probably in orienting and straightening the microtubular array GBP and Dsh are released at the opposite point of sperm entry Future Dorsal side of embryo They inactivate GSK3 allowing -catenin accumulation Ventral side GSK3 is degraded
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The Dorsal Signal: -Catenin
7.20 The role of Wnt pathway proteins in dorsal-ventral axis specification, -catenin (orange)
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Function of the Organizer
Nieuwkoop center cells remain endodermal, cells of the organizer become dorsal mesoderm and migrate underneath dorsal ectoderm Dorsal mesoderm induces central nervous system formation Properties of organizer – 4 major functions Ability to self differentiate dorsal mesoderm Ability to dorsalize surrounding mesoderm into paraxial mesoderm (somites) Ability to dorsalize ectoderm forming neural tube Ability to initiate the movement of gastrulation
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Function of the Organizer
Organizer cells ultimately contribute to 4 cell types: Pharyngeal endoderm Head mesoderm (prechordal plate) Dorsal mesoderm (primarily notochord) Dorsal blastopore lip 1 & 2 – induce forebrain and midbrain 3 – induces hindbrain and trunk 4 – remains to end of gastrulation then becomes chordaneural hinge that induces the tip of the tail
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Induction of Neural Ectoderm and Dorsal Mesoderm: BMP Inhibitors
Problem: There is NO molecule secreted by the organizer and received by the ectoderm How is neural tissue made from ectoderm? Organizer – ectoderm – neural tissue NO! It is the epidermis induced to form and NOT the neural tissue Ectoderm is induced to become epidermal tissue by binding to Bone Morphogenetic Proteins (BMPs)
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Induction of Neural Ectoderm and Dorsal Mesoderm: BMP Inhibitors
Nervous system forms from region of ectoderm that is protected from epidermal induction (BMP) By BMP-inhibiting molecules The “default fate” of ectoderm is to become neural tissue Certain parts of embryo induce the ectoderm to become epidermal tissue by secreting BMPs The organizer tissue acts by secreting molecules that block BMPs – thus allowing ectoderm “protected” by BMP inhibitors to become neural tissue
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Regional Specificity of Neural Induction
Regional differences Regional specificity of neural structures Forebrain Hindbrain Spinocaudal region Organizers not only form neural tissue but also specify regions Can be induced
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Regional Specificity of Neural Induction
Figure Regional and temporal specificity of induction
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Specify Left Right Axis
Most parts symmetrical Heart and internal organs are not Xenopus gene Xnr1 Xenopus nodal related 1 Expression of nodal gene in the lateral plate mesoderm on the left side of embryo Determine heart and gut side Clockwise rotation of cilia keeps it to the left side Pitx2 activated by Xnr1 Normally expressed on left side and controls which side heart and gut folds occur 7.36 Pitx2 determines the direction of heart looping and gut coiling
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