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Chapter 07-Amphibians & Fish Early development and axis formation. 7.1 Formation of the parallel arrays of microtubules extend along the vegetal hemisphere along the future dorsal-ventral axis. These microtubules allow the cortical cytoplasm to rotate with respect to the inner cytoplasm.

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slide1

Chapter 07-Amphibians & Fish

Early development and

axis formation

slide2
7.1 Formation of the parallel arrays of microtubules extend along the vegetal hemisphere along the future dorsal-ventral axis

These microtubules allow the cortical cytoplasm to rotate with respect to the inner cytoplasm

7 1c reorganization of cytoplasm in the newly fertilized frog egg part 1
7.1C Reorganization of cytoplasm in the newly fertilized frog egg (Part 1)

animal

Gastruation will begin in the gray crescent, region

opposite of sperm entry

(Ventral )

Dorsal

First cleavage (80%)

Vegetal

figure 10 3 cleavage of a frog egg
Figure 10.3 Cleavage of a Frog Egg

Cleavage in amphibians

The first division begins at the animal pole and slowly extends down cleavage

Figure 10.3A

4 micromeres:

quick division

  • Two major functions:
  • Permit cell migration
  • Prevents the contact of the vegetal cells destines to become endoderm with those cells fated to give rise to the skin and nerves

4 macromeres:

quick division

The vegetal hemisphere ultimately contains larger and fewer blastomeres than the animal half.

or “size discrepancy”

Figure 7.2,3 Scanning Electron Micrographs of the Cleavage of a Frog Egg

figure 7 4 depletion of ep cadherin mrna in the xenopus oocyte
Figure 7.4 Depletion of EP-cadherin mRNA in the Xenopus Oocyte

While the cells are dividing in the oocyte, numberous cell adhesion molecules keep the blastomeres together. EP-cadherin: a maternal supply

Antisense oligonucleotides

Control

The adhesion between the blastomeres is dramatically reduced, resulting in the obliteration of the blastocoel

slide8
Figure 10.25 Model of the Mechanism by which the Disheveled Protein Stabilizesb-catenin in the Dorsal Portion of the Amphibian Egg

Translocated

dorsally

released

(GSK-3b+Axin1(2)+APC) complex for b-catenin degradation

Dsh protein is the inhibitor of GSK-3b

slide9
10.24 Model of the mechanism by which the Disheveled protein stabilizes -catenin in the dorsal portion of the amphibian egg (Part 1)
slide10
10.24 Model of the mechanism by which the Disheveled protein stabilizes -catenin in the dorsal portion of the amphibian egg (Part 2)
slide11

b-Catenin

Tcf/Lef

Tcf/Lef

Tcf/Lef

Transcription

(c-Jun,fra-1,c-myc

& cyclin D1 etc.)

The Wnt signaling pathway in normal and cancer development

Wnt

Fz(frizzled)

DSH

GBP

Akt

GSk-3b

GSk-3b

GSk-3b

APC

APC

APCmt

Axin

b-Catenin

Axin

Axinmt

P

P

b-Catenin

b-Catenin

b-Catenin

P

Oncogenic

b-Catenin

P

Stablization

degradation

b-Catenin

b-Catenin

n

n

b-Catenin

CBP

ICAT

Groucho/TLE

Chibby

Repression

Transcription

(c-Jun,fra-1,c-myc

& cyclin D1 etc.)

Embryonic cell

Differentiated cell

Tumor cell

a maternal supply cytoplasmic determinants and cell cell signals in cell differentiation

Cytoplasmic determinants in the egg.The unfertilized egg cell has molecules in its cytoplasm,

encoded by the mother’s genes, that influence development. Many of these cytoplasmic

determinants, like the two shown here, are unevenly distributed in the egg. After fertilization

and mitotic division, the cell nuclei of the embryo are exposed to different sets of cytoplasmic

determinants and, as a result, express different genes.

a Maternal supply: Cytoplasmic Determinants and Cell-Cell Signals in Cell Differentiation

Cytoplasmic determinants in the cytoplasm of the unfertilized egg

Regulate the expression of genes in the zygote that affect the developmental fate of embryonic cells

Unfertilized egg cell

Sperm

Molecules of

another cyto-

plasmic deter-

minant

Molecules of a

a cytoplasmic

determinant

Fertilization

Nucleus

Sperm

Zygote

(fertilized egg)

Mitotic cell division

Two-celled

embryo

figure 7 5 fate maps of the blastula of the frog xenopus laevis
Figure 7.5 Fate Maps of the Blastula of the Frog Xenopus laevis

Amphibians Gastrulation

The three germ layers

Fate mapping has shown that cells of the Xenopus blastula have different fates depending on whether they are located in the deep or

The superfical layers. The mesodermal precursors exist mostly in the deep layer of cells (the internal cytoplasm around the equator), while the ectoderm (the animal hemisphere surface) and endoderm (the vegetal hemisphere surface) arise from the superficial layer on the surface.

Animal

equator

Vegetal

figure 7 6 cell movements during frog gastrulation

early

Mid-

endoderm

Figure 7.6 Cell Movements During Frog Gastrulation

(primitive gut)

Sperm entry

Fig. 10.8

Dorsal

Marginal

Zone cells

Figure 7.7 Surface View of An Early Dorsal Blastopore Lip of Xenopus

slide17

10.9 A graft of cells from the dorsal marginal zone of a salamander embryo sinks into a layer of endodermal cells and forms a blastopore-like groove

figure 7 14 spemann s demonstration of nuclear equivalence in newt cleavage
Figure 7.14 Spemann’s Demonstration of Nuclear Equivalence in Newt Cleavage

Organizer for axis formation

Spemann’s experiment I

His baby

girl’s hair

Spemann’s experiment: early amphibian nuclei were geneticall identical and that each cell was able to give rise to an entire animal.

Early amphibian nuclei were genetically identical and that each cell was capable of giving rise to an entire organism

figure 7 15 asymmetry in the amphibian egg
Figure 7.15 Asymmetry in the Amphibian Egg

Spemann’s experiment II:

Only the blastomere containing the gray crescent develops normally

The gary crescent region gives rise to the cells that initiate gastrulation. These cells form the dorsal lip of the blastopore.

Fig. 10-2

The movements of fertilized amphibian eggs expose a gray crescent shaped are of cytoplasm in the region directly opposite the sperm entry point.

No dorsal

structures

figure 10 18 determination of ectoderm during newt gastrulation

(interspcies transplantation)

Figure 10.18 Determination of Ectoderm During Newt Gastrulation

Spemann’s experiment III:

He found the cells of the early gastrula were uncommitted (regulative/conditional development), but the fates of late gastrula cells were determined(autonomous/independent/mosaic development).

可朔性高

uncommitted

X

可朔性低

autonomous

Why ?

Organizer

organization of a secondary axis by dorsal blastopore lip tissue
Organization of a Secondary Axis by Dorsal Blastopore Lip Tissue

Spemann’s ( and Mangold) experiment IV:interspcies transplantation-Triturus taeniatus (pigmented; donor) vs Triturus cristatus (nonpigmented; host) .

They found that all the tissue in the early gastrula, only one has its fate determined. It is the dorsal blastopore lip tissue (derived from the gray crescent cytoplasm)-the Organizer

slide35
Figure 10.10 Twin Blastopores Produced by Rotating Dejellied Xenopus Eggs Ventral Side Up at the Time of First Cleavage

Although the sperm entry is not needed to induce the movements in the egg cytoplasm.

The centrifuged eggs and two dorsal blastopore lips formed, leading to the formation of twin larvae

Also see in zebrafish

Siamese

slide38
Figure 7.18 Summary of Experiments by Nieuwkoop and by Nakamura and Takasaki, Showing Mesodermal Induction by Vegetal Endoderm

Mechanisms of axis determination-Nieuwkoop center; b-catenin

The experiments of

Pieter Nieukoop and Osamu Nakamura

Removed

b-catenin

The signal (TGF-b & FGFs) induces the marginal cells to become mesoderm

figure 10 21 transplantation experiments on 64 cell amphibian embryos
Figure 10.21 Transplantation Experiments on 64-cell Amphibian Embryos

Nieuwkoop center demonstration

The transplantation experiments of Gimlich & Gerhart (1984)

blastopore

The three vegetal blastomeres opposite the point of sperm entry are able to indurce the formation of the dorsal lip of the blastopore and a complete dorsal axis.

also see

Fig. 10-23

slide41

Figure 10.23 The Regional Specificity of Mesoderm Iinduction Can Be Demonstrated by Recombining Blastomeres of 32-Cell Xenopus Embryos (also see Fig. 10-11, 22C)

Sperm

entrance

The dorsalmost vegetal cells induced the animal pole cells to become dorsal mesoderm

figure 10 24 the role of wnt pathway proteins in dorsal ventral axis specification
Figure 10.24 The Role of Wnt Pathway Proteins in Dorsal-Ventral Axis Specification

Molecular biology of the Nieuwkoop center

Molecular pathway of

The major candidate for the factor that forms the Nieuwkoop centerin the dorsalmost vegetal cells isb-catenin

b-catenin

2-cell

b-cat is a key player in the formation of the dorsal axis.

Knockdown:lack of dorsal structure

Overexpression: 2nd axis

Wnt signaling pathway-key molecue

Dorsal side

/nuclear localization b-catenin

Also see in zebrafish

Siamese

ventral side

/ no nuclear localization b-catenin

gastrula

Dominant

negative GFK-3b

slide43
Figure 7.21 Model of the mechanism by which the Disheveled protein stabilizes β-catenin in the dorsal portion of the amphibian egg

(GSK-3b+Axin1(2)+APC) complex for b-catenin degradation

Dsh protein is the inhibitor of GSK-3b

slide44

Figure 7.21 Model of the mechanism by which the Disheveled protein stabilizes β-catenin in the dorsal portion of the amphibian egg (Part 1)

slide45
7.22 Summary of events hypothesized to bring about the induction of the organizer in the dorsal mesoderm

Figure 7.23

Nuclear

localization

Goosecoid can activate genes whose proteins are responsible for organizer’s activities

Siamois and Xlim1, function together to activate the goosecoid gene in the organizer

slide47
Figure 7.23 Mesoderm Induction and Organizer Formation by the Interaction of b-catenin And TGF-b Proteins

intermediate Xnrs

Later mesoderm

high Xnrs

Low Xnrs

Ventral

mesoderm

+

gradient

Figure 10.20C

Mesoderm-inducing signals

figure 10 28 ability of goosecoid mrna to induce a new axis

control

Two dorsal lips

Two dorsal axis

Twin embryos

Figure 10.28 Ability of goosecoid mRNA to Induce a New Axis

Organizer-specific transcription factors

Two diifusible proteins:

BMP inhibitors → b-Cat↑

(Ventralization)

Wnt inhibitors → b-Cat ↓

(Dorsalization)

BMP

Wnt

BMP

Functions of the organizer:

Dorsal mesoderm

Paraxial mesoderm

Dorsallized ectoderm (neural tube)

Initation of gastrulation

Wnt

BMP

BMP

Wnt

BMP

figure 7 25 rescue of dorsal structures by noggin protein
Figure 7.25 Rescue of Dorsal Structures by Noggin Protein

Noggin

1. It induced dorsal ectoderm to form neural tissue.

2. It dorsalized medoderm cells that would otherwise become ventral mesoderm.

3. Inhibition of BMP2&4.

UV damaged Xenopus eggs

Noggin

mRNA

b-catenine

Dorsal zone

Dorsal

lip

notochord

Figure 10.31 Localization of Noggin mRNA in the Organizer Tissue,Shown by In Situ Hybridization

figure 10 32 localization of chordin mrna
Figure 10.32 Localization of Chordin mRNA

Chordin

It dorsalized mesoderm and induction of CNS

Inhibition of BMP2&4.

figure 7 27 model for the action of the organizer
Figure 7.27 Model for the action of the organizer

BMP4 vs. Organizer

Bone morphogenesis protein 4 (BMP4) is a powerful ventralizing factor.

It was known that an antagonistic relationship between BMP4 and the organizer. If the mRNA of BMP4 is injected into xenopus eggs, all the mesoderm in the embryo becomes ventrolateral mesodrm, and no involution occurs at the blastopore lip.

Overexpression of a dominant negative BMP4 receptor resulted in the formation of two dorsal axes.

figure 10 34 cerberus mrna injected into a single d4 blastomere

Extra head was derivaed form cerberus mRNA injected I to a single D4 (ventral vegetal

Blastomere of a 32-cell embryo.

Figure 10.34 Cerberus mRNA injected into a Single D4 Blastomere

Wnt inhibitors

Frzb, is a small & solube form of Frizzed, the Wnt receptor.

Dickkopf also directly interact with the Wnt receptor

Cerberus promotes the formation of the head

structures (the cement gland, eyes, and olfactory placodes.

Inhibitor for

neural induction

Figure 10.36 Xwnt8 Is Capable of Ventralizing the Mesoderm and Preventing Anterior Head Formation in the Ectoderm

Inhibition of trunk formation form frzb mRNA injected I to the marginal zone.

Figure 10.35 Paracrine Factors From the Organizer are Able to BlockCertain Other Paracrine Factors

Figure 10.37 Frzb Expression and Function

slide53

Figure 10.34 Regional Specificity of Induction can be Demonstrated by Implanting Different Regions (Color) of the Archenteron Roof into Early Triturus Gastrulae

Regional specificity of Induction

Forebrain, hindbrin, and spinocaudal regions of the neural tube must all be properly organized

in an anterior-to posterior direction.

Late grastrula

early grastrula

forebrain

hindbrain

tail

Figure 10.35 Regionally Specific Inducing Action of the Dorsal Blastopore Lip

Two transplantation expeiments show that the first cells of the organnizer to eneter the embryo induce the formation of head structures, while those cells that form the dorsal lip of later-stage become spinal cords and tail.

figure 10 42 the wnt signaling pathway and posteriorization of the neural tube

b-catenin gradient

Figure 10.42 The Wnt Signaling Pathway And Posteriorization of the Neural Tube

The posterior transforming proteins:Wnts

The primary protein involved in posteriorizing the neural tube

is a member of the Wnt family of paracrine factors, Xwnt8

Overexpression

(Wnt inhibitor)

(Wnt )

P

hindbrain

midbrain

posteriorization

anteriorization

forebrain

A

Wnt/b-catenin

gradient

For AP axis

BMP gradient

For DV axis

b-catenin

figure 10 44 organizer function and axis specification in the xenopus gastrula
Figure 10.44 Organizer Function and Axis Specification in the Xenopus Gastrula

Dorsalization

/posteriorization

anteriorization

slide59

Figure 11.2 Screening Protocol for Identifying Mutations of Zebrafish Development

ENU (N-ethyl-nitro-ureal) mutagenesis

figure 11 4 discoidal meroblastic cleavage in a zebrafish egg
Figure 11.4 Discoidal Meroblastic Cleavage in a Zebrafish Egg

45’

15’

Blastoderm

meroblastic/

discoidal

fish blastula
Fish Blastula

10 cell division (mid-blastula):three cell populations

(EVL)

The most superficial cells above

The blastoderm;

Extraembryonic protection

(YSL): a ‘ringof nuclei’ region;

Some cell movemments

fish blastula1
Fish Blastula

Fate mapping:

Cells in specific regions of the embryo give rise to certain tissues in a highly predictable manner.

figure 11 6 2 cell movements during gastrulation of the zebrafish
Figure 11.6(2) Cell Movements During Gastrulation of the Zebrafish

The embryonic shield is functionally equivalent

to the dorsal blastopore lip or organizer of amphibians, since it can organize a secondary embryonic axis when transplanted to a host embryo

(Figure 11.8).

figure 11 6 3 cell movements during gastrulation of the zebrafish
Figure 11.6(3) Cell Movements During Gastrulation of the Zebrafish

90% epiboly

(9 hours)

Completion of gastrulation

(10.3 h; first somite

Mesendoderm

Endoderm

slide70

07-09 Zebrafish Gastrulation 1.mpg

07-10 Zebrafish Neurulation.mpg

07-11 Zebrafish Cell Movements.mpg

figure 7 45 the embryonic shield as organizer in the fish embryo
Figure 7.45 The Embryonic Shield as Organizer in the Fish Embryo

Two axes formed,

Sharing a common

Yolk cell

In the embryo that had had its embryonic shield removed, no dorsal structures formed, and the embryo lacked a nervous system

figure 11 9 axis formation in the zebrafish embryo
Figure 11.9 Axis formation in the Zebrafish Embryo

Zwnt8

Nieuwkoop center

In both fish and amphibians, BMPs and certain Wnt proteins made in the ventral and lateral regions of the embryo would normally induce the ectoderm to become epidermis

The precursors of these two regions are responsible for inducing the ectoderm to become neural ectoderm

Xwnt8

amphibians

figure 11 10 b catenin activates organizer genes in the zebrafish
Figure 11.10 B-Catenin Activates Organizer Genes in the Zebrafish

b-catenin nuclear localization

zebrafish

Future shield

shield

b-catenin

Xenopus

Nieuwkoop center

(Siamois)

These proteins that block BMPs and Wnts and allow the specification of the dorsal Mesoderm and neural ectoderm

+ zTcf3

Anterior movements

& dorsal convergent extension

slide74

Specifying the Left-right Axis

A nodal, Xnr1→pix2

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