Bio 120 2005 Lecture 9

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Bio 120 2005 Lecture 9

• Cloning implications
• Insect development

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human reproductive cloning

• basic biology is likely to be similar --we are
  mammals
• but low success rates pose a problem
• USA no federally funded research
• UK, Canada restricted, but allowed
• Germany total ban

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Proposed outcomes (if it ever worked)

• Vanity cloning
• eternal life? but your clone would be even less
  similar than an identical twin
• Eugenics, Dysgenics
• clone Einstein, Hitler, armies
• Sentimental cloning
• clone dead child, parent, pet
• all the above rest on wrongheaded genetic
  determinism, i.e., that DNA determines identity.
• cloning is not photocopying
• Infertility treatment?
• there are easier ways...

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therapeutic cloning

• Goal is to make genetically matched ES cells
• Proof-of-concept in mice
• allows somatic (not germline) gene/cell therapy

Rideout WM et al. 2002. Correction of a genetic
defect by nuclear transplantation and combined
cell and gene therapy. Cell 109 17-27.
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therapeutic cloning

• goal is to make ES cells
• ethical worries are with use of blastocysts
• isnt there any other way to convert regular
  cells into ES cells?
• not yet
• adult stem cells very controversial, and not as
  pluripotent as ES

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therapeutic cloning questions

• what makes blastocysts special
• biologically?
• ethically? why are other cells not so
  privileged?
• they are potential individuals
• but any cell is

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The ethical debate

• Kant humans are an end in themselves, not a
  means
• Bentham the greatest good for the greatest
  number
• is a zygote an individual? totipotency means any
  cell is a potential individual!

Immanuel Kant
Jeremy Bentham
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The questions

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

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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

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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
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Insects
Wingless insects (e.g. silverfish)
Hemimetabolous insects Incomplete
metamorphosis (dragonflies, bugs, roaches,
earwigs, lice)
Holometabolous insects Complete
metamorphosis (beetles, butterflies, wasps, flies)
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Drosophila life cycle
Fig 2.29
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Drosophila development
http//flymove.uni-muenster.de/ Movies of
development and anatomy Interactive animations
of genetic mutants e.g. Processes/segmentation/
Highly recommended
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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
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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/

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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
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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

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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
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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
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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)
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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

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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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Friedrich Seidel (1897-1992)
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2. 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

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Cytoplasmic 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

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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

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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?

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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

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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

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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

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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
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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
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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
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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

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Gradient explanation of gaps
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Gradient explanation of cytoplasmic transfers
Wait a couple of hours
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