The First Steps into Embryo Development:

Cleavage, Gastrulation, and Germ Layer Formation

Xenopus two-cell stage

www.nikonsmallworld.com

LSM 3233 Developmental Biology of Animals Lecture 2 (23 Aug 2013)

Xenopus early blastula late blastula

Q&A
- Put into the correct temporal order:
Acrosome Reaction Cortical Reaction Cortical Rotation Capacitation

- What is correct?
Germ cells are continuously produced. Male or female? All germ cells are already formed at birth. Male or female?

- You plan a cell transplantation experiment in early stage embryos. Which model organism would you choose?
Mouse Chicken Xenopus Zebrafish

Re-cap from last week:

Fertilization results in calcium waves and the opening of cortical granules: Cortical Reaction

Ca2+ increase leads to fusion of cortical granules with membrane: release of enzymes (proteases, hexoaminidases) digest ZP3 and change ZP structure prevent further binding and penetration of spermatozoa (zona reaction)

Taken from Inoue N. et al., Nature, 2005

The animal-vegetal axis of Xenopus is maternally determined

One end of the egg (top) is the animal pole (pigmented) whilst the other (bottom) is the vegetal pole (pale) Animal pole contains the nucleus Vegetal pole contains the yolk Planes of early cleavages is defined by the animal-vegetal axis

Cytoplasmic movement in the frog egg after fertilization Cortical rotation

future dorsal

http://biology.kenyon.edu

Gray crescent Region of intermediate pigmentation in the marginal zone of the amphibian egg caused by a shift in the pigmented egg cortex toward the site of sperm entry; marks the future site of the dorsal lip of the blastopore

Reorganization of cytoplasm in the newly fertilized frog egg

Single sperm enters at any site on the animal hemisphere. Following fertilization, there is a 30 shift of the cortical cytoplasm with respect to the inner cytoplasm. Gray crescent always forms opposite to sperm entry point (gives rise to the future dorsal side of the embryo). The shifting of cytoplasm moves morphogenetic determinants to certain areas of the embryo that will be important later in establishing the future dorsal-ventral axis during development.

Gilbert SF Fig. 5-33

The principle of assymetric localization of local determinants

Vg1 mRNA in Xenopus oocyte

Alberts et al., 2002 Fig. 21-66

Unequal distribution of maternal RNAs and proteins with important roles in embryonic axis formation

RNAs

Vg1 encodes a secreted growth factor of the TGF family; mRNA localized in vegetal half of oocyte VegT encodes a T-box containing transcription factor; mRNA localized in vegetal half of oocyte

Other localized maternal determinants:

Proteins

-catenin acts as cell adhesion molecule, but also as nuclear transcription factor (localized dorsally after cortical rotation) Dishevelled (Dsh) interacts with -catenin and prevents its degradation

The Wnt signalling pathway

In development
Wnt
binds its receptor frizzled activates Dishevelled prevents GSK mediated degradation of -catenin -catenin translocates to nucleus binds to LEF/TCF transcription factor activates target genes

and in cancer

important for:
organizer formation mesoderm induction neural induction and patterning bone development

Mutations in APC (Adenomatous polyposis colon) or -catenin lead to constitutive nuclear translocation and cancer

The first cell divisions during Cleavage Phase result in an asymmetric distribution of determinants to daughter cells

Alberts et al., 2002 Fig. 21-66

Significance of cleavage
www.ohsu.edu

Cleavage subdivides zygote into many blastomeres; each with a certain inheritance. The nucleus to cytoplasm ratio increases; i.e. cell division without corresponding cell growth. Rapid mitotic divisions of the embryos require specific proteins such as histones, tubulins,and RNA polymerases, which are provided by maternal stocks of proteins and mRNAs.
med.unsw.edu.au

When a frog embryo is divided into halves at the 4-cell stage, the half that did not contain a region underneath the gray crescent, called the Nieuwkoop center, develops abnormally

Nieuwkoop Center is specified by cortical rotation

belly piece

Wolpert L. et al., 2002 2nd ed

Pieter Nieuwkoop (1917-1996): Discovered the mechanisms of embryonic axis formation

Methods to interfere with Neuwkoop Center (NC) activity: Egg rotation can mimic the effect of sperm entry (GRAVITY!)

Blastopore

also: - UV-irridiation: leads to degradation of Neuwkoop center no axis - LiCl treatment: ectopic activation of Neuwkoop center dosalization

Transplantation of Nieuwkoop Center cells rescues ventralized embryos classic modern

belly piece

dorsalizing substance Kalthoff, K., 2nd ed Fig. 9.15

DeRobertis et al., Nat Rev Genet 1, 2000

The dorso-ventral axis of amphibian embryos is determined by the site of sperm entry

Cortical rotation of the egg cytoplasm results in the formation of a signaling center (Nieuwkoop Center) in the vegetal region (gray crescent!) Nieuwkoop Center defines the site of onset of gastrulation (dorsal blastopore lip; Spemann organizer)

blastopore
Wolpert L. et al., 2002 2nd ed

The different stages of embryonic development

Fertilization

Growth and maturation Birth Organogenesis Neurulation

10,000 cells 30,000 cells

Cleavage period

Gastrulation

Morphogenesis

It is not birth, marriage or death which is the most important time in your life, but gastrulation
Lewis Wolpert, 1989

Gastrulation leads to germ layer formation and mesoderm induction

Three germ layers give raise to all organs of the body, plus the germ line. These germ layers are formed during gastrulation.
Gilbert SF 2006, Fig. 1.1

This is only a selection of tissues formed!

Gastrulation
Rearrangement of cell layers by migration and division resulting in the formation of the three embryonic germ layers: ectoderm, endoderm, and mesoderm

Wolpert L. et al 2nd ed., 2002

Fate map

Fate

Gastrulation and Germ Layer Formation

What is gastrulation? What is the significance of gastrulation? Why is the dorsal blastopore lip called the organizer? How do axes get defined in the early vertebrate embryo? How do germ layers get established?
Drosophila
www.molbio1.princeton.edu/wieschaus/

Gastrulation in frogs
Dorsal lip/ Blastopore

Epiboly

Involution

Extension

IMZ = involuting marginal zone

Gilbert SF 2006, Figs. 10.9, 10.13

Xenopus and zebrafish: Different modes of cleavage

2-cell 1.5 h

2-cell 0.75 h

stage 8 5.2 h Xenopus Holoblastic cleavage (complete)

1000-cell 3.3 h Fish Meroblastic cleavage (incomplete)

Gastrulation in fish Dome stage:

onset of gastrulation by yolk pushing

Epiboly
cell migration around yolk sac

Involution
cells change direction of migration; happens around complete margin Embryonic shield = Dorsal lip convergence/extension cells migrate from ventral to dorsal

The three morphogenetic cell movements during zebrafish gastrulation

Epiboly

Involution

Convergence

Migrating cells change the direction of their paths Spatial re-organization of cell layers

The dorsal lip is an embryonic Organizer

Wolpert L. et al., 2002 2nd ed

H. Mangold and H. Spemann (1924) called this process primary induction The dorsal blastopore lip was named organizer

Timing matters: the organizer changes its activity over time

Wolpert L. et al 2nd ed., 2002

Early Ectopic head

Late: Ectopic tail

Organizer activity is also found in other vertebrates: The Hensons node in chicken
Primary induction works across species barrier (here quail vs. chick or duck)

Hensons node = Spemann organizer = Shield

Organizer formation
What defines the position of the organizer (dorsal blastopore lip) and the onset of gastrulation?

The Nieuwkoop Center forms first, and defines the position of the Spemann organizer

Spemann organizer arises just above the Nieuwkoop Center during the late blastula-early gastrula stage Signals from the Spemann organizer are involved in further patterning along both the dorso-ventral and anterior-posterior axes of the embryo, as well as inducing the CNS.

What are the molecular components of the Nieuwkoop center?

Maternal factors cooperate to set up the Nieuwkoop Center

Vg1 in Xenopus oocyte

Gilbert SF Fig. 5-33

Gilbert SF Fig. 10-26

Nuclear localization of -catenin exclusively on the dorsal embryo side

dorsal view

ventral view

Summary (so far)

an external signal (sperm entry) sets the dorsoventral axis Cortical rotation causes rearrangement of cytoplasmic determinants A synergism of these determinants leads to the establishment of the Nieuwkoop center, which in turn induces the organizer This induction defines the position of gastrulation and sets up the organizer (triggers formation of the three germ layers)

The role of the organizer: To trigger formation of the germ layers

All tissues of the body are derived from three germ layers endoderm, mesoderm, ectoderm

Induction and patterning of the mesoderm

Fate map of the frog embryo (at blastula)

Specification map Shows fate of a blastula cell when isolated and placed in culture Fate map Shows normal fate of a blastula cell in the embryo

Why are the two maps different? Because cells in the embryo are under influence of signals from neighboring cells and tissues.

Where do the signals determining cell fate come from?

Nieuwkoops experiments:
Signals from the vegetal pole - are responsible for mesoderm induction - lead to stabilization of a dorsal determinant (Nieuwkoop center) opposite to the sperm entry point - establish the organizer at the future dorsal side
Wolpert L. et al., 2002 2nd ed

Evidence that at least two signals come from the vegetal region

muscle, notochord

blood
Wolpert L. et al., 2002 2nd ed

Mesoderm induction: The Three signal model

Signals are needed to tell cells
- to become mesoderm - to differentiate into a distinct subtype of mesoderm

The Three signal model of mesoderm induction

1. Tissue recombinations tell us that vegetal tissue can induce mesoderm from animal cap ectoderm. 2. Nieuwkoop showed that dorsal vegetal tissue (DV) induced the organizer. 3. The organizer secretes signals that instruct formation of dorsal mesoderm derivatives (notochord). Possible other signals: Ventralizing! Wnt8, BMPs
(Bone Morphogenetic protein) instruct formation of ventral mesoderm

The molecular nature of the Nieuwkoop Center

siamois: Homeobox transcription factor

VegT (secreted) induces nodal growth factors (Xnrs) VegT with -catenin induces higher level of Xnrs -catenin induces siamois Xnrs (high) with siamois induce organizer Organizer secrets factors that antagonize ventral BMP (important for mesoderm patterning and neural induction)

-catenin together with high nodal concentrations induce expression of organizer genes

Gilbert SF Fig. 10-26

Dorsal stabilization of -catenin

Activin

Gilbert SF, 2006 Fig. 10-25

dorsal

ventral

Early steps are controlled by maternal factors. Later:

Zygotic gene expression of mesoderm specifying factors in Xenopus at late blastula

Expression domains of a number of zygotic genes encoding transcription factors correspond quite well to demarcations on the specification map

Brachyury mutant zebrafish (no-tail)

Does the Three-signal model also work in other vertebrates? Xenopus

zebrafish

Gilbert SF 2006, Fig. 11.7

Stabilized nuclear -catenin on the dorsal side of both Xenopus and zebrafish embryos

Similar, yet not identical signaling factors regulate mesoderm formation in different vertebrates

Is this everything?

From Kimelman D, Nat Rev Genet 7, 2006

Summary:
Mesoderm induction in Xenopus
vegetal region
signal 1 (general inducing signal) e.g. Vg-1, VegT

induction

signal 2: -catenin; high Xnr, siamois dorsal mesoderm with Spemann organizer signal 3: dorsalizing signals from organizer (e.g. noggin, chordin)

ventral mesoderm also: ventralizing signal low Xnr, BMP2/4, Xwnt-8

patterning of mesoderm
(blood, muscle, notochord)

Outlook:
Neural induction and patterning
epidermal neural

www.luc.edu/faculty/wwasser/dev/midneur.gif

Next lecture: Fri, 30 Aug