Formation of body axes in vertebrates: frogs and birds

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Lecture 3

• Formation of body axes in vertebrates frogs and
  birds

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where do asymmetries come from?

• Animal-vegetal asymmetry of oocyte
• environmental asymmetry e.g. sperm
• random symmetry breaking

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Amphibians

• Early work (Spemann, et al.) on newts (urodeles),
  or anurans (e.g. Rana)
• Since 1950s, Xenopus laevis, the African clawed
  frog
• Very easy to rear in lab (although tendency to
  escape)
• Ovulation inducible by injection of chorionic
  gonadotrophin
• frog or toad? no difference
• Minor differences in embryogenesis between
  anurans (frogs) and urodeles

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Fate map of frog oocyte
An
Ectoderm (epidermis, nervous system)
Mesoderm (notochord, somites, blood, heart,
kidney)
Endoderm (gut)
Could there be germ-layer determinants already
asymmetric in oocyte?
Vg
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sperm entry breaks symmetry

• George Newport (1854)
• Gastrulation always starts opposite SEP
• Sperm entry point (SEP) defines future ventral
  side
• First cleavage plane bisects SEP and animal pole
  meridional

.
SEP
animal pole
Blastopore initiates
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Cortical rotation
one big cell

• In some amphibians (Rana, but not Xenopus), a
  gray crescent develops opposite the SEP
• Due to relative rotation of outer cortex (dark)
  to inner endoplasm
• Rotation shown in Xenopus by marking core with
  dye spots, hold cortex fixed

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Cortical rotation is aligned with the sperm entry
point
(Complex mixing of endoplasm--not totally
understood) W Fig 3.3, 3.6
meridian of maximal rotation passes through
animal pole and SEP, defines plane of first
cleavage
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By the 4-cell stage dorsal and ventral sides are
different
W Fig 3.4
sperm entry point future ventral
future dorsal side
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Experimental evidence for causal relationships

• Hypothesis cortical rotation defines the
  dorso-ventral axis.
• Three kinds of evidence
• Correlation show it
• Loss-of-function block it
• Gain-of-function move it
• Cf. Kochs postulates for causative role of
  pathogen
• sometimes hard to get all 3 kind of evidence

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1. show it

• cortical rotation occurs after sperm entry and
  before 1st cleavage--right time
• direction of cortical rotation correlates with
  sperm entry and dorsal axis formation

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cortical rotation and microtubules (MTs)

• MTs in shear zone between cortex and endoplasm
  in vegetal half
• sperm centrosome is microtubule minus-end
  organizing center
• does cortical rotation require MTs?

before fertilization random
after fertilization polarized with minus ends at
sperm centrosome
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2. block it

• Are MTs required for rotation/axis formation?
• Treat embryos with nocodazole or UV light to
  disrupt MT polymerization
• block rotation, ventralize embryo dose-dependent
  
• Therefore (1) MTs required for rotation (2)
  rotation required for dorsalization

Ventralized embryo no gastrulation, no dorsal
tissues
UV
(Wolpert page 73)
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3. move it

• Tipping rescues effects of UV irradiation, and
  can even over-ride normal rotation
• Works because endoplasm is heavier than animal
  cytoplasm, slips to bottom, rotates with
  respect to cortex
• Therefore rotation is sufficient to dorsalize

no tipping
UV
Tip by 90 for 30 min
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evidence

• Does cortical rotation set up the dorsoventral
  axis?
• Correlation yes, present at right time, place
• Loss-of-function yes, blocking rotation (UV)
  blocks dorsalization
• Gain-of-function yes, artificial
  (gravity-induced) rotation can rescue UV block

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The Nieuwkoop center
define by dorsalizing activity of blastomeres in
transplants
W Fig 3.5
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The Nieuwkoop center

• center of dorsalizing activity in dorsal
  vegetal (future endoderm) blastomeres
• Major role is to induce the Spemann organizer in
  dorsal marginal cells (future mesoderm)
• (1) How is it formed?
• (2) How does it signal?

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dorsal cytoplasm contains dorsalizing activity
move it by cytoplasmic transfer
block it by removing vegetal pole
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dorsalizing activity can be detected at 1-cell
stage

• Transplants of cytoplasm as bioassay
• Dorsalizing activity initially vegetal, then
  shifts dorsally during cortical rotation
• what is the activity in molecular terms?

microtubule- dependent
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Candidates for the dorsalizing activity

• Several mRNAs localized to vegetal pole of
  oocyte Vg1, XWnt-8 (growth factors).
• Translated after fertilization proteins become
  concentrated in dorsal cells
• Look at one in detail beta-catenin

Bright dots indicate Vg1 mRNAs, detected by in
situ hybridization
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beta-catenin and dorsalizing activity

• Show it b-catenin protein is enriched dorsally
• Block it inhibition of b -cat by antisense RNA
  injection causes ventralization
• Move it injecting b -catenin sense mRNA into
  ventral vegetal cells causes second axis

W Fig 3.8
b-catenin is necessary and sufficient for
dorsalizing activity how is it enriched on dorsal
side??
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Lithium causes dorsalization
Picture from Essential Developmental Biology by
Slack
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Why is Lithium dorsalizing?

• b-catenin is initially in all cells, then
• Degraded ventrally, stabilized dorsally, so only
  accumulates in dorsal nuclei
• GSK-3 degrades b-catenin
• Lithium inhibits GSK-3, allowing b-catenin to be
  stabilized everywhere--the dorsal fate
• what is the endogenous inhibitor of GSK-3?

W Fig 3.9
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How does cortical rotation lead to dorsal
stabilization of b-catenin?

• Current best model Dishevelled (Dsh) locally
  inhibits GSK3
• Dsh translocates from vegetal pole to dorsal side
• microtubule-based transport
• dorsal inhibition of GSK3 leads to stabilization
  of b-catenin
• Miller et al 1999

endoplasm is labeled with Nile red
dye Dishevelled protein is tagged with Green
Fluorescent protein (GFP)
future dorsal side
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What are the inductive signals from the Nieuwkoop
center?
animal

• Combination of dorsal determinant (b-catenin)
  and vegetal determinant (Vg1 or VgT) leads to
  activation of Xnr (Xenopus nodal-related) signal
  only in dorsal vegetal cells
• Xnrs activate genes (siamois) in dorsal marginal
  cells

dorsal
vegetal
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axis formation is self-organizing

• activation
• pricking egg with needle mimics some events
  caused by fertilization
• Ca influx
• cortex rotates in random direction
• therefore sperm entry orients cortical rotation
  but is not essential for it to occur

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DV axis in frogs

• Oocyte polarity (An/Vg) prefigures the germ
  layers, but not the body axes
• Dorsoventral axis defined by cortical rotation
  sperm entry breaks symmetry but not required for
  rotation itself
• Cortical rotation rearranges cytoplasm, resulting
  in the dorsalizing Nieuwkoop center in dorsal
  vegetal cells
• b-catenin stabilized dorsally by local inhibition
  of GSK3
• Nieuwkoop center later induces the gastrula stage
  Spemann organizer in dorsal marginal cells

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Bird (chick) development

• extremely large eggs
• Cleavage confined to blastoderm (2 mm)
• discoidal cleavage, generates blastodisc of
  60,000 cells at time of laying
• best vertebrate embryo for studying organogenesis

W Fig 2.10
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development in utero

• Oocyte grows in ovary
• Released into oviduct, where fertilized
• Travels down oviduct (24h) where coated with
  albumen, shell, etc
• Cleavage starts 5h after fertilization
• blastodisc has

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Chick cleavageformation of epiblast and
hypoblast

• epiblast will give rise to embryo
• hypoblast makes extra-embryonic tissues (needed
  for prolonged development in eggshell)

W Fig 2.12
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Chick gastrulation
W Fig 2.13

• Epiblast cells migrate into subgerminal space to
  become mesoderm, endoderm
• primitive streak thickened region where this
  happens equivalent to blastopore of frog
• Defines future dorsal midline of body

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Chick organogenesis
W Fig 2.14

• Hensens node regresses (moves anterior to
  posterior)
• somites form from anterior to posterior
• analogous to frog except on a disc, not in a
  sphere

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Polarization
Area opaca (thick, darker)
Marginal zone
Area pellucida (thin, transparent)

• Blastoderm initally appears symmetrical
• First asymmetry (about time of laying) is a
  thickening of area pellucida called Kollers
  sickle adjacent to the posterior marginal zone
  (PMZ)
• Primitive streak develops from sickle within 16h
  of egg-laying

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Von Baers Rule

• if an egg is horizontal with the pointed end to
  the right, the tail will be towards the observer

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Gravity polarizes the blastodisc

• Egg turns on long axis 200 times in
  oviduct/uterus
• yolk is heavy, does not rotate but tilted
• Side of blastoderm uppermost in gravity field
  will become PMZ

W Fig 3.10
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evidence for gravity as asymmetric cue

• remove early egg from oviduct and suspend in
  oblique orientation--PMZ always on top
• Critical period 14-16 h after fertilization
• if suspend horizontally in saline, PMZ forms at
  random
• Gravity biases but is not essential for
  polarization
• Mechanism? Unclear, but must be happening at
  multicellular stage (unlike gravity tipping in
  frog)

Kochav and Eyal-Giladi, 1971
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The PMZ can organize a primitive streak

• W Fig 3.11 (Eyal-Giladi Khaner 1989)
• transplanted PMZ can sometimes induce second
  streak
• But usually the older (bigger) streak inhibits
  development of other streaks
• Example of lateral inhibition

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a PMZ inhibits rest of disc from making PMZ
_
_
example of lateral inhibition
W 1.14
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the blastodisc is highly regulative

• Divide blastodisc into 4, each piece makes
  embryo (cf. Driesch)
• regulation--any part of disc can make a PMZ
• problem for disc is to prevent multiple PMZs
  (twinning)

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More evidence for lateral inhibition
_
_

• Recombine FOUR quarter-discs (each with half a
  PMZ) into one
• Regulation--only one primitive streak

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but lateral inhibition has its limits
Combine four HALF-discs into one
_
_
_
_
disc is double normal size
twinning
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Summarize chick

• Entire blastodisc has potential to form embryo,
  raising developmental problem of twinning
• Gravity provides a bias so that one region gets a
  head start to form PMZ
• PMZ inhibits rest of blastodisc from doing same
  (lateral inhibition molecular mechanism
  uncertain but may involve hypoblast signals)
• Result one embryo per egg

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Similarities and differences between frog and
chick

• Nieuwkoop center and PMZ both form at dorsal
  midline in future posterior
• Both induce organizers in dorsal mesoderm
  (Spemann organizer, Hensens node)
• Lithium dorsalizes frog blastula and chick
  epiblast
• BUT frog axis generated in 1-celled zygote,
  chick axis in multi-cellular blastodisc

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Axis formation

• polarization by self-organizing processes that
  ensure robust asymmetries
• External cues (sperm entry, gravity) can bias
  these axis-forming processes, but are not
  essential
• Earliest asymmetries are crude. Evolutionarily
  flexible.
• How do mammals do it? Next lecture