Cleavage, Blastula & Gastrulation in Vertebrates

Cleavage, Blastula & Gastrulation in Vertebrates

Chapters 10, 11

I. Amphibians

•  Cleavage

•  Gastrulation

•  Axis Formation

I A. Cleavage

•  Holoblastic radial

•  Mesolecithal

•  Telolecithal

•  Heavy yolk in vegetal hemisphere slows cleavage rate to 1/50th rate in animal hemisphere

•  1st cleavage meridional, slower thru vegetal half

•  2nd cleavage meridional, perpendicular to 1st

–  Begins before 1st cleavage completed

•  3rd equatorial, offset towards animal hemisphere (yolk effect)

•  As cleavage progresses, animal hemisphere divides more rapidly than vegetal hemisphere

•  Smaller blastomeres (micromeres) in animal hemisphere

•  Morula: 16 to 64 cell stage

•  Blastula: 128 cell stage

–  Clear blastocoel, offset towards animal hemisphere

•  Blastomeres held together with CAM’s

•  EP-cadherin is critical for blastomere adhesion

–  Made from maternal mRNA in cytoplasm

•  If block translation with antisense RNA, embryo “falls apart”

“Things fall apart; the centre cannot hold; Mere anarchy is loosed upon the world.”

•  might have been written about the embryo’s need for CAM’s during development.

•  What is the source and who is the author of the quote?

“And what rough beast, its hour come round at last, Slouches towards Bethlehem to be born?”

•  If slouching implies the anterior-posterior axis did not differentiate correctly, the above quote would describe an animal with mutations in what genes?

Mid-blastula transition

•  Change over from stored mRNA to newly synthesized mRNA

•  In Xenopus, new transcription begins late in 12th cell cycle

I B. Gastrulation

Xenopus as model system, some variability in other amphibian species

•  Cells move with invagination, involution, and epiboly

•  Fate Map (pre-gastrulation)

–  Ectoderm and endoderm superficial

–  Mesoderm under surface in equatorial (marginal) zone (in some species mesoderm superficial)

I B 1. Cell Movements in Gastrulation

•  Invagination of endoderm “bottle cells” just below equator in gray crescent

–  Bottle cells narrow at surface, long neck, thicker at base

–  Marginal zone

•  Involution of marginal zone cells

–  Dorsal lip

–  First prechordal plate (head mesoderm)

–  Then chordamesoderm (notochord)

•  Extension of blastopore laterally and ventrally

–  Movement thru bottle cell invagination and marginal zone involution

–  Moves endoderm and mesoderm internal

•  Extension of blastopore laterally and ventrally

–  Yolk plug shows through blastopore

•  Epiboly of surface ectoderm

–  Moves towards blastopore

–  Covers surface of embryo

•  (mesoderm also involutes in illustration)

I B 2. Localizing Blastopore

•  Forms opposite point of sperm entry

•  Rotation of cortical cytoplasm towards sperm entry point

–  Allows contact of vegetal pole cortex proteins with deeper cytoplasm at point opposite sperm entry

•  Sperm entry point = ventral side

•  Side opposite sperm entry = dorsal side

•  Cytoplasmic rotation brings together different regions of cytoplasm

•  The cells that receive these cytoplasms are vegetal

•  In Xenopus, 3 vegetal blastomeres of the 64 cell embryo can induce blastopore formation

•  These three cells use β-catenin as “dorsal signal”

•  UV-irradiated zygote does NOT gastrulate

•  However, transplanted vegetal blastomere restores ability of irradiated embryo to gastrulate

•  Transplant ventral blastomere to normal embryo

•  Get dual sites of gastrulation

I C. Axis Formation

Progressive determination due to cellular interaction with surrounding cells

•  Hans Spemann experiments

•  Primary Embryonic Induction (Spemann and Mangold)

•  Mechanism of Axis Formation

•  Regional Specificity of Induction

I C 1. Hans Spemann Experiments

Nuclear Equivalence

•  Tie off embryo at 1st cleavage, keep nuclei on one side

•  At 16 nuclei stage, allow 1 nucleus to go to other side

•  Twins developed

Gray Crescent

•  If constriction did not include gray crescent in both halves, only half with gray crescent developed

•  Half without gray crescent “Bauchstück”, a “bellypiece”

Narrowing of Cell Fate

•  Transplant cells in early and late gastrula

•  Early gastrula: cells differentiate in accordance with NEW location (cell fate changed)

•  Late gastrula: cells differentiate in accordance with ORIGIN (cell fate NOT changed, already determined)

I C 2. Primary Embryonic Induction

•  Spemann and Hilde Mangold

•  Transplanted dorsal blastopore lip from early gastrula to another gastrula

•  Induced second axis of development

–  Conjoined twins

•  Dorsal lip organizes new axis of development

•  Thus, dorsal lip and derivatives (notochord, prechordal mesoderm) set up sequence of inductive events

I C 3. Mechanism of Axis Formation

•  Nieuwkoop Center

•  Functions of Organizer

•  Ventralizing Gradient

•  Regional Specificity of Induction

I C 3 a. Nieuwkoop Center

•  Sperm penetration causes cytoplasmic rotation towards sperm entry point

–  Sperm entry point à ventral

–  Opposite side à blastopore à dorsal

•  Cortex rotation brings disheveled protein from vegetal pole “up” to point opposite sperm entry

•  This area now has enrichment of disheveled protein

•  Disheveled protein prevents degradation of β-catenin in dorsal-most endoderm cells

–  This region is Nieuwkoop Center

•  Nieuwkoop Center induces mesoderm above N.C. to become “organizer”, the blastopore dorsal lip

•  Inject antisense RNA to β-catenin

–  No dorsal structures

•  Inject β-catenin in ventral side embryo

–  Second axis of development induced

•  What does β-catenin do?

•  Is transcription factor, results in production of goosecoid

•  Goosecoid activates genes in Spemann organizer (dorsal lip)

•  Goosecoid injections can induce second site of gastrulation (second organizer)

•  Disheveled à β-catenin à goosecoid à Organizer

I C 3 b. Functions of Organizer

•  Organizer is dorsal mesoderm

•  Migrates via involution

•  Spreads in midline between ectoderm and endoderm

Has five major functions

•  Initiate gastrulation

•  Become dorsal mesoderm (chordamesoderm)

•  “dorsalize” surrounding mesoderm to become lateral mesoderm

•  “dorsalize” overlying ectoderm into neuralectoderm

•  Convert neural ectoderm into neural tube

• 

•  LiCl treatment increases conversion of organizer mesoderm

•  Increases area where goosecoid produced

•  Dorsalizes embryo

•  What did LiCl do in sea urchins?

–  What molecule was influenced by LiCl?

I C 3 c. Ventralizing Gradient

•  BMP-4

•  Binds to ectodermal cells

•  Where binds get epidermal cells (BMP-4 induces epidermal cells)

•  No binding, get neuralectoderm cells

•  Organizer determines whether or not BMP-4 binds to ectoderm

•  Organizer secretes proteins (e.g., noggin, chordin, follistatin, sonic hedgehodge, nodal-related proteins) that bind to BMP-4 to prevent BMP-4 binding to receptors

•  Where BMP-4 does NOT bind (due to organizer effect), cells become neuralectoderm

•  Thus, “induction” of neuralectoderm by Organizer occurs by blocking normal BMP-4 signal that converts ectoderm to epidermal ectoderm

BMP-4 has two main effects

•  Converts ectoderm to epidermal ectoderm

•  “Ventralizes” mesoderm (Kidney, blood, muscles)

I C 3 d. Regional Specificity of Induction

•  Organizer mesoderm not only induces neural tube (blocks BMP-4), but specifies regions along anterior/posterior axis

Otto Mangold (1933)

•  Transplanted different regions of archenteron roof of late gastrula

•  Different regions induced different ectodermal derivatives

–  Anterior à balancers + oral ectoderm

–  Next region posterior à head (eyes, nose)

–  Next region posterior à hindbrain

–  Next region posterior à doral trunk, tail

•  Molecular basis due to “dual gradient”

–  Anterior-transforming: highest anterior, causes anterior structures

•  Chordin, Noggin, Follistatin, Nodal-related (Organizer-derived)

•  IGF

–  Posterior-transforming: highest posterior, causes posterior structures

•  eFGF, Retinoic Acid, Wnt

II. Fish

•  Cleavage

•  Gastrulation

•  Axis Formation

II A. Cleavage

•  Meroblastic, discoidal

•  Macrolecithal

•  Telolecithal

•  Cleavage restricted to blastodisc

•  1st 12 divisions synchronous

•  10th division onset mid-blastula transition

II B. Gastrulation

•  Yolk cells underneath cleaved area are endoderm

•  Cleaved area separates into

–  Upper epiblast

–  Lower hypoblast (involution and/or ingression)

•  Embryonic shield site of mesoderm movement (like dorsal lip of frog blastopore)

•  Epiblast (ectoderm) spreads via epiboly

II C. Axis Formation

•  Similar to amphibians

•  Embryonic shield acts like frog dorsal lip

–  BMP and Wnt proteins

•  Embryonic shield acts like frog dorsal lip

–  Transplant of embryonic shield to embryo induces second axis of development

•  Embryonic shield induced by a “Nieuwkoop Center”, involving β-catenin

Amniote Development

•  Birds, reptiles, mammals

•  Develop amnion (extraembryonic membrane)

•  Are differences in cleavage patterns

Cleavage:

•  Birds & reptiles: meroblastic cleavage

–  Macrolecithal, telolecithal

•  Mammals: rotational cleavage

–  Microlecithal, isolecithal

•  However, all 3 share gastrulation patterns

Gastrulation:

•  Same basic pattern in all three groups

•  Pattern in birds and reptiles “influenced” by yolk

•  Pattern in mammals follows pattern of birds and reptiles, despite mammalian “low yolk” egg

III. Bird

•  Cleavage

•  Gastrulation

•  Axis Formation

III A. Bird Cleavage

•  Telolecithal

•  Macrolecithal

•  Meroblastic, discoidal

–  Cleavage limited to small disc-shaped, yolk-free area at animal pole (2-3 mm diameter)

–  Cleavage furrows animal-vegetal axis (“meridional” or vertical), do not meet or enter yolk

–  Later horizontal furrows separate blastomeres from yolk

•  Blastoderm cells linked via tight junctions

•  Space under cells subgerminal space

•  Blastoderm divided into two areas

–  Outer Area opaca, in contact with yolk

–  Inner Area pellucida, separated from yolk by subgerminal space

•  Marginal zone a thin layer of cells between area opaca and area pellucida

III B 1. Bird Gastrulation: Epiblast and Hypoblast

•  Blastoderm delamination

•  Cells move into subgerminal space

•  Form primary hypoblast (polyinvagination islands)

•  Cells from posterior marginal zone (Koller’s sickle) migrate anteriorly

•  Fuse with primary hypoblast to form secondary hypoblast

•  Double layered embryo

–  Upper epiblast à embryo and amnion, chorion, allantois

–  Lower hypoblast à yolk sac

–  Blastocoel between epiblast & hypoblast

III B 2. Bird Gastrulation: Primitive Streak & Cell Movements

•  Primitive streak forms at posterior edge epiblast as thickening

•  Due to

–  Ingression of endodermal cells into blastocoel

–  Migration of lateral cells to median

•  Primitive streak elongates anteriorly (60-70% of area pellucida length)

•  Secondary hypoblast moving anteriorly at same time

•  Primitive streak defines body axes:

–  Anterior/posterior

–  Left/Right

•  Primitive groove forms in streak

•  Opening for cell migration inward via ingression

–  Mesoderm in middle

–  Endoderm deeper, displace hypoblast

•  Hensen’s Node (primitive knot) at anterior edge

•  Cells migrate over HN anteriorly

–  Foregut

–  Head mesoderm

–  Notochord

•  Hensen’s Node named for Victor Hensen

•  Hensen’s node equivalent to amphibian dorsal lip

•  What happens if Hensen’s node transplanted to 2nd embryo?

•  Primitive streak now regresses

•  Hensen’s node migrates posteriorly

•  As Hensen’s node migrates posteriorly anterior region undergoes neurulation and other organogenesis

•  Thus, anterior development is advanced over posterior development

•  Gastrulation continues

•  Mesoderm moves down thru primitive groove and laterally

•  Endoderm moves down thru primitive groove deep and laterally

•  Chordamesoderm migrates in over Hensen’s node and migrates anteriorly

•  Ectodermal cells spread via epiboly over all of egg (including yolk)

•  Takes about 4 days to enclose egg

III C. Bird Axis Formation

•  Posterior Marginal Zone (PMZ) induces formation of primitive streak

•  PMZ transplanted to anterior region à induce primitive streak and Hensen’s node

•  PMZ like Nieuwkoop center

–  β-catenin localization

–  Induces Hensen’s node formation

Hensen’s node

•  Composed of epiblast and Koller’s sickle cells

•  Equivalent of amphibian dorsal lip

–  Site of gastrulation

–  Cells become chordamesoderm

–  Transplant to another embryo à 2nd axis development

–  Secretes chordin, noggin, nodal proteins, goosecoid

IV. Mammalian

•  Cleavage

•  Gastrulation

•  Axis Formation

IV A. Mammalian Cleavage

•  Secondary oocyte released from ovary

•  Picked up in oviduct

•  Fertilized in oviduct

•  Meiosis completed

•  Cleavage begins 24 hours after fertilization

•  Subsequent early cleavage divisions every 12-24 hours

•  New transcription in mouse as early as 2-cell stage

•  Microlecithal

•  Isolecithal

•  Holoblastic, rotational

–  1st cleavage meridional

–  2nd cleavage

•  Meridional in one cell

•  Equatorial in other cell

–  Asynchronous, odd numbers of blastomeres

Human Development

IV A. Mammalian Cleavage

•  Compaction at 8 cell stage

•  Cell adhesion increases, embryo becomes more compact

•  Cell contact increases

•  Tight junction between outer cells

–  Become trophoblast

•  Gap junctions between inner cells

–  Become ICM/ES cells

•  Cavity forms within embryo à blastocyst stage

•  Blastocyst digests way out of zona pellucida (with strypsin)

•  Implants in endometrium

IV B. Mammalian Gastrulation

•  ICM delaminates into

–  Upper epiblast

–  Lower hypoblast

•  Epiblast splits into

–  Upper amniotic layer

–  Lower embryonic epiblast

•  Primitive streak and Hensen’s node forms at posterior end epiblast

•  Migrates anterior, then regresses

Cells ingress:

•  Anterior from H.N.

•  Laterally for mesoderm

•  Down to hypoblast for endoderm

IV C. Mammalian Anterior/Posterior Axis Formation

•  Two main signaling centers

–  Hensen’s node

–  Anterior Visceral Endoderm (AVE)

•  Hensen’s Node

–  Creation of body, secretes chordin and noggin

•  AVE

–  Along with HN, responsible for head formation

Hox Genes

•  Homologous to HomC (homeotic complex) of Drosophila

•  HomC

–  Two gene clusters (Antennapedia, Bithorax)

–  Chromosome #3

–  Genes arranged in order of expression & association with body segment specialization

Hox Genes

•  4 copies of Hox complex per haploid genome

•  Each copy (a, b, c, d) on different chromosome

•  Hox = mouse; HOX = human

•  Paralogous groups: similar genes in the 4 Hox complexes (e.g., a1, b1, d1)