Title: Bio 120 2006 Lecture 9
1Bio 120 2006 Lecture 9
- Insect development and morphogens
2The questions
- How are body axes set up?
- How are germ layers specified
- How are germ layers subdivided and patterned?
3Arthropods
- 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
4The 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
5Insects
Wingless insects (e.g. silverfish)
Hemimetabolous insects Incomplete
metamorphosis (dragonflies, bugs, roaches,
earwigs, lice)
Holometabolous insects Complete
metamorphosis (beetles, butterflies, wasps, flies)
6Drosophila life cycle
Fig 2.29
7Drosophila development
http//flymove.uni-muenster.de/ Movies of
development and anatomy Interactive animations
of genetic mutants e.g. Processes/segmentation/
Highly recommended
8The 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
9Cleavage 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/
10Gastrulation
- 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
11The 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
12long-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
13The 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
14Today 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)
15Experimental 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
161. 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)
172. Ligature experiments
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- 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
18Cytoplasmic transfer experiments
(1)
(2)
2 hours
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(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
19Conclusions
- 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
20Two 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?
21Morphogenetic 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
22Response 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
23Fields
- 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
24The 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
25Response 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
26How 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
27Simple 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
28Gradient explanation of gaps
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29Gradient explanation of cytoplasmic transfers
Wait a couple of hours
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30The 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
31look 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
32Overview
- Hierarchy of regulatory genes
- Signaling centers established during oogenesis
- Mutations display maternal effects
- Zygotic genes turn on after fertilization
- Segmentation and making segments different
33Zygotic 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)
34Maternal 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
35Four 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
36Thoughts 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?