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Bio 120 2006 Lecture 9

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How are germ layers subdivided and patterned? Arthropods. Hardened exoskeleton, articulated body segments, Jointed appendages. probably' monophyletic ... – PowerPoint PPT presentation

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Title: Bio 120 2006 Lecture 9


1
Bio 120 2006 Lecture 9
  • Insect development and morphogens

2
The questions
  • How are body axes set up?
  • How are germ layers specified
  • How are germ layers subdivided and patterned?

3
Arthropods
  • Hardened exoskeleton, articulated body segments,
    Jointed appendages
  • probably monophyletic
  • gt 1 million described species, estimated 10-30
    million
  • Outnumber humans by ratio of 108 1
  • Subphylum Uniramia
  • Class Myriapoda centipedes, millipedes
  • Class Insecta insects
  • Other subphyla Crustacea, Chelicerates

4
The insect body plan
  • Three body regions
  • head (5-6 segments)
  • thorax (3)
  • abdomen (8-11)
  • body is segmented unsegmented terminal regions
  • 3 pairs of legs (on the three thoracic
    segments)-Hexapoda

m ? f
5
Insects
Wingless insects (e.g. silverfish)
Hemimetabolous insects Incomplete
metamorphosis (dragonflies, bugs, roaches,
earwigs, lice)
Holometabolous insects Complete
metamorphosis (beetles, butterflies, wasps, flies)
6
Drosophila life cycle
Fig 2.29
7
Drosophila development
http//flymove.uni-muenster.de/ Movies of
development and anatomy Interactive animations
of genetic mutants e.g. Processes/segmentation/
Highly recommended
8
The Drosophila egg
  • About 0.5 mm long
  • Yolk in center
  • Visible AP and DV asymmetry before fertilization
  • eggshell (chorion) is also polarized--made by
    follicle cells
  • Sperm entry via micropyle (little gate) in
    eggshell

dorsal
A
P
ventral
9
Cleavage and cellularization
Fig 2.30
Tubulin actin
Tubulin myosin
  • nuclei divide every 9 min without cytokinesis
  • Cellularization all at once
  • Movies from Bill Sullivans lab
    http//bio.research.ucsc.edu/people/sullivan/

10
Gastrulation
  • Ventral blastoderm invaginates to form mesoderm
    (muscles etc)
  • Anterior, posterior invaginations form gut

Cross sections of embryos immunostained for
Twist, a bHLH protein expressed in mesoderm
Fig 2.31
11
The germ band
  • Ventral blastoderm, after gastrulation, will give
    rise to most of embryo
  • Undergoes extension then retraction
  • segmentation first visible during extended germ
    band stage (top)
  • embryonic units are parasegments, different from
    larval segments

12
long-germ versus short-germ insects
  • Drosophila (and other advanced insects)
    Long-germ development
  • germ band develops from most of blastoderm
  • Segments form simultaneously
  • Beetles ( other primitive insects) Short or
    intermediate germ development
  • Part of blastoderm first forms anterior segments
  • Posterior segments develop progressively from
    growth zone (cf. somitogenesis)
  • different routes to similar extended germ band
    stages

Fig 5.34
13
The Drosophila larva
  • Feeding machine
  • Segmented, obvious AP and DV pattern in cuticle
  • Pupates, larval tissues self-destruct (autolysis)
  • Adult (imago) rises from the ashes via imaginal
    discs

Figs 2.33, 2.34
14
Today axis formation
  • 1. Experimental embryology suggests that simple
    mosaic models are not enough
  • 2. Morphogen gradient models of pattern
    formation
  • 3. Using genetics in Drosophila to identify the
    morphogens

The red-banded leaf-hopper (not Euscelis)
15
Experimental embryology of insects
  • Leaf hoppers short-horned bugs (Hemimetabola)
  • Euscelis incisus (formerly E. plebejus)
  • Intermediate-germ development
  • Large eggs, soft egg shell, amenable to
    manipulations
  • Endosymbiotic bacteria in posterior
  • See section 5.18

16
1. evidence for a posterior organizer
  • Suck out tiny bit of cytoplasm from posterior
  • Lose thorax and abdominal segments
  • Long-range effect the activation center

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12
Friedrich Seidel (1897-1992)
17
2. Ligature experiments
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1 2 7 8 9
  • Klaus Sander (1950s) ligate egg with thread
  • lose segments in middle of pattern
  • remaining segments in correct order, spread out
  • Earlier ligature gives bigger gap

18
Cytoplasmic transfer experiments
(1)
(2)
2 hours
123456789
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9 8 7 7 8 9
9 8 7 7 8 9
(same experiment as Fig 5.36)
  • Move posterior cytoplasm by poking with needle
  • ligate immediately--posterior bicaudal,
    anterior makes nothing
  • same experiment except wait 2 hours before
    ligating
  • now anterior forms complete pattern

19
Conclusions
  • Posterior cytoplasm is special
  • Probably nothing to do with the bacteria, these
    just a convenient marker
  • Rest of egg highly regulative
  • Long-range effects, not explained by simple
    mosaic model
  • Sanders model diffusible morphogen made in
    posterior
  • Also independence of A-P and D-V axes

20
Two questions of pattern formation
  • How can cell fate be determined by relative
    position? (what is the positional information)
  • How can a cells response vary depending on its
    history?

21
Morphogenetic fields
shoulder
limb
X
  • Newt limb development (Spemann)
  • Remove limb disc, limb flank regulates
  • Disk flank constitute a developmental field
    region in which cell fate determined by relative
    position

22
Response to signals depends on history (I.e.
genome)
  • Spemann Schotte 1932
  • Transplant newt ventral cells into frog gastrula,
    newt teeth where frog mouth (no teeth) should be
  • cells fates were appropriate for their position
    and for their ancestry

23
Fields
  • Embryonic territories that communicate to form a
    structure
  • E.g. the limb field etc
  • Cells in a field are equivalent in developmental
    potential (at first)
  • Cells become different in response to signals
  • Signals produced from signaling centers, and have
    concentration-dependent effects

24
The French Flag analogy
  • Flag area field
  • Cells read out position relative to boundary
    (flagpole)
  • Response depends on
  • Local concentration of morphogen relative to
    threshold values
  • Cells own history

Fig 1.22
25
Response depends on history (genotype)
  • Cells are newt (UK) or frog (French) in genotype
  • Both respond to same signals
  • Response (UK or French) depends on history

(newt mouth)
(frog gastrula)
Fig 10.36
26
How do you get gradients?
  • Localized source of morphogen that can diffuse
    over gt1 cell diameter
  • Morphogen must be unstable if degraded
    everywhere, a dispersed sink
  • Exponential decay gradient from source
  • Localized source/Dispersed sink (LSDS) model
  • Gradient could form over small (gt 1 mm)
    territories in 1 hour given known diffusion
    constants

morphogen
Distance from source
27
Simple LSDS models
  • Can explain
  • Why organizers can pattern large groups of
    cells--because they are morphogen sources
  • How ordered patterns form--because morphogen
    gradient has polarity
  • defect regulation--why pattern reforms if small
    bits of field removed or added (because source
    still intact)
  • Have difficulty explaining
  • Size invariance (e.g. Dictyostelium)
  • Ability of sources to re-form (e.g. limb field)
  • And do not address
  • how cells can read out local morphogen
    concentrations
  • Box 10A reaction-diffusion models that
    self-organize

28
Gradient explanation of gaps
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1 2 7 8 9
29
Gradient explanation of cytoplasmic transfers
Wait a couple of hours
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9 8 7 7 8 9
30
The awesome power of genetics
  • Christiane Nusslein Volhard, Eric Wieschaus,
    Trudi Schupbach, Ruth Lehmann, Kathryn Anderson
  • Nobel Prize for Physiology or Medicine 1995
  • Systematic hunts for fly mutants with embryonic
    patterning defects (1977-1987)
  • First screens looked for zygotic mutations
  • Second round looked for maternal effect mutations
    (more difficult)
  • Key was large scale--find all the genes involved

31
look for mutants with abnormal cuticle pattern
  • Cuticle has obvious AP and DV patterns
  • Mutagenize flies with chemical mutagen
  • Screen for pattern defects in F2, F3 generation

D V
Dark-field LM
Scanning EM
32
Overview
  • Hierarchy of regulatory genes
  • Signaling centers established during oogenesis
  • Mutations display maternal effects
  • Zygotic genes turn on after fertilization
  • Segmentation and making segments different

33
Zygotic mutations
  • Phenotype depends on zygotic genotype
  • gene is expresed in zygote

m/ F
m/ M
X
m/m, m/, /
25 of F1 progeny are phenotypically Mutant (if
recessive)
34
Maternal effect mutations
  • Phenotype depends on mothers genotype, not on
    zygote genotype
  • Seen if gene product (RNA, protein) is made by
    mother and placed in oocyte

m/
m/
X
Fm/m not mutant
/ m
X
Heterozygotes are mutant, but mutation is not
dominant--it is recessive with maternal effect
m/ 100 mutant
35
Four classes of maternal-effect phenotypes
1 2 3 4 5 6 7 8 9
9 8 7 5 6 7 8 9
12 3 4
2 3 4 5 6 7 8
Fig 5.3
  • Also dorsoventral mutants

36
Thoughts from screens
  • Number of genes is in 10s not 100s
  • Problem is tractable
  • Some phenotypes resemble Sanders experiments
  • Genes may affect equivalent signaling centers
  • independence of AP and DV axes
  • Four phenotypic classes, each defined by multiple
    genes
  • 4 genetic pathways?
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