2006Lecture 11

1
2006-Lecture 11

• Drosophila dorsoventral axis
• segmentation

2
First 2 signals in DV axis
DORSAL
Follicle cell epithelium
oocyte
torpedo
oocyte
1. Dorsalizing Signal Grk-Top
grk
oocyte
nuc
Perivitelline (extracellular) space
Toll
2. Ventralizing Signal Spz-Toll
spz
VENTRAL
3
After Toll is activated
Fig 5.8
4
The dorsoventral pattern of fates
dorsal

• 1. amnioserosa (extra-embryonic, surrounds yolk)
• 2. dorsal ectoderm (epidermis)
• 3. ventral ectoderm (neurectoderm)
• 4. mesoderm (will invaginate)

ventral
5
3. zygotic readout of DV polarity

• gradient of DL protein in ventral half turns on
  various genes, represses dpp
• in dorsal half, dpp expressed
• lateral cells express sog

Fig 5.14
6
protein gradients along DV axis
Fig 5.15

• countergradients of DPP and SOG (not simple LSDS)
• DPP BMP-like, SOG chordin-like

7
Do insects and vertebrates use the same mechanism
to pattern DV axes?

• Vertebrate (Xenopus)
• BMP ventralizes
• noggin/chordin dorsalize by inhibiting BMP
• Insect (Drosophila)
• DPP dorsalizes
• SOG dorsalizes by inhibiting DPP
• Xenopus noggin can dorsalize fly embryo!
• convergent or divergent evolution? if divergent,
  how come the gradients are inverted?

8
The Cuvier-Geoffroy debate
Geoffroy, inverted dissection of lobster 1822

• Georges Cuvier animals belong to four
  divisions (vertebrates, articulates, molluscs,
  radiates) that are fundamentally different

Erienne Geoffroy St. Hilaire note the
similarity between body plan of lobster and
vertebrate if one is inverted
See section 15.7
9
Geoffroy may have been right (after 170 years)
DPP

• similarity BMP inhibition on side that forms
  CNS
• difference is in relative position of mouth
• from blastopore in insects (protostome)
• forms secondarily on other side in vertebrates
  (deuterostome)

insect
blue CNS red CV system
Inhibitor (SOG)
vertebrate
chordin
sog
inverted insect
BMP-4
DPP
Figure 15.18
10
Development of AP axis

• Segmentation
• division into regular repeating units (metameres)
• Segment diversification
• making segments different--next lecture
• two processes are independent but must be
  coordinated

11
Models for periodic patterns in space

• Clock and wavefront
• progressive segmentation
• somitogenesis
• Reaction-diffusion models
• self-organizing standing waves
• stripes of pigmentation (zebra etc)

12
segmentation genes

• Nusslein-Volhard and Wieschaus, 1980
• screens for zygotic pattern mutants
• 30 genes, 3 classes, forming spatial hierarchy
• GAP
• PAIR-RULE
• SEGMENT POLARITY

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phenotypic hierarchy

• gap mutants
• lack multiple adjacent segments
• other segments form OK
• pair-rule mutants
• lack alternating segments
• have 7 double-wide stripes (instead of 14)
• segment polarity mutants
• every segment missing same part of pattern
• since 1980s genes cloned and regulation studied
  at molecular level

14
The gap genes

• 7 genes hunchback, Kruppel, giant
• transcriptionally activated by maternal proteins
• encode txn factors
• proteins unstable, form transient concentration
  gradients

15
the gap proteins are local morphogens

• hunchback protein in gradient (thanks to bcd,
  nos)
• high HB represses Kr
• medium HB activates Kr
• low HB has no effect
• result Kr txn turns on only in single thin
  stripe!
• how did they figure it out? by altering HB gene
  dosage and looking at Kr expression

Fig 5.18
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cross-inhibition sharpens gap domains

• gradients overlap at first
• each gap gene activates itself and represses
  others
• result each level of AP axis has unique
  combination of gap proteins at blastoderm stage
• each gap domain spans several segments--how are
  specific segments defined?

an example of lateral inhibition
17
the pair-rule genes

• gt8 genes even-skipped (eve), fushi tarazu
  (ftz), etc
• txn turned on by gap proteins before
  cellularization
• encode transcription factors
• expressed in alternating parasegments (PS), not
  segments

Fig 5.21
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Parasegments

• first overt signs of segmentation (at extended
  germ band) are pits in ectoderm
• originally thought to correspond to segment
  boundaries
• in fact out of register--segment boundaries form
  later, between pits
• embryonic repeating unit named the parasegment

19
Animations from scanning EMs by Thom Kaufman
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pair-rule gene expression is dynamic

• example even-skipped
• stage 10 expression low, uniform
• stage 14 (cellularization) 7 distinct stripes
• stripes initially fuzzy, then sharpen anterior
  borders (refinement)--involves autoactivation

one parasegment
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how do we get from non-periodic gap domains to
periodic pair-rule patterns?

• eve stripe 2 (PS3)
• combinatorial control by gap proteins
• BCD and HB activate
• GT, KR repress
• equivalent mechanisms for other stripes

Fig 5.22
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combinatorial control of transcription involves
binding to enhancer regions
eve

• the eve stripe 2 enhancer
• 600 bp regulatory element in DNA of gene
• many binding sites for gap proteins
• note e.g. GT binding site overlaps HB site

activators
repressors
Fig 5.23
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evidence that stripes are made piecemeal
see the LacZ or GFP turn on in these patterns
make flies expressing these transgenes
2
1
3
4
5
6
7
reporter (LacZ, GFP)
1. eve regulatory DNA
1
3
4
5
6
7
2.
2
3.
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getting from 2-parasegment to 1-parasegment
stripes
first

• each stripe is made independently, by local
  combination of gap proteins (no standing wave)
• enhancer regions integrate combinatorial inputs
• cross-regulation so that overlapping 2-segment
  stripes yield 1-segment stripes (4 cells wide)

eve
ftz
then
PS
PS
PS
PS
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segmentation the next stage

• pair-rule genes define transient boundaries of
  parasegments
• embryo then cellularizes
• cells must maintain memory of boundaries--role of
  the segment polarity genes
• but first--when do segments become specified?

26
when are cells determined to form specific
segments?

• first, need to examine when segments allocated
• fate mapping using genetic markers clonal
  analysis

27
mitotic recombination
m
m
1.

m
Xray
m
m
2.




see Box 5B also
28
use genetic markers to see clones

• Clone group of mutant cells descended from
  single mutant daughter
• homozygous for genetic marker--stable,
  cell-autonomous, does not dilute, e.g. multiple
  wing hairs (mwh)
• irregular shape reflects local cell mixing

non-mwh cells
mwh clone
29
cells allocate to segments at cellularization
early clone

• induce clone before cellularization--crosses
  segment borders
• after cellularization, stays within segment
  (multiple tissues)
• (caveat--later clones are smaller, but even when
  made large by Minute technique they never cross
  the line)
• additional transplant experiments showed cells
  are determined at cellularization

late clone
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The discovery of compartments

• Antonio Garcia-Bellido, Pedro Ripoll and Gines
  Morata (1973)
• make clones in wing imaginal disc
• make big clones using Minute trick (Box 5B)
• clones never cross an invisible line straight
  down the middle of the wing
• Model wing divided into anterior and posterior
  compartments (lineage units)

Fig 5.26
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engrailed and compartments

• in engrailed mutant wings, clones behave as if
  there is no invisible line
• posterior wing looks like anterior (margin hairs)
  P ?A

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Where is engrailed required?

• make engrailed mutant clones in wild-type (en/)
  wing
• clones in anterior still respect boundary (I.e.
  en is not required in anterior)
• clones made in posterior do not!
• conclusion engrailed required only in posterior
  cells to make them different from anterior cells

anterior
en/en
en/en
en/
posterior
33
why do segments have 2 compartments?

• compartment boundaries are the relics of the
  parasegment boundaries formed in the embryo
• initially defined by sharp anterior domains of
  eve or ftz expression
• engrailed is transcribed only in cells with high
  levels of eve or ftz

Fig 5.25
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engrailed

• homeodomain protein
• expressed in anterior 2/3 of every parasegment
• the anterior part of each parasegment forms the
  posterior compartment of the mature segment

engrailed gene fused to GFP
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segment polarity mutants

• within each segment
• anterior 1/3 has rows of denticles (hairs)
• posterior 2/3 naked
• segment polarity mutants disrupt pattern in every
  segment
• gt20 genes, variety of phenotypes

parasegment
wild type
a segment polarity mutant transforms posterior
(naked) into anterior (hairy)--hence hedgehog,
armadillo, gooseberry etc
Fig 5.30
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Local signals at parasegment boundaries

• engrailed
• expressed in cells posterior to boundary
• activates its own transcription and that of
  hedgehog (hh, secreted)
• hh signaling activates wingless
• wingless (wg)
• expressed anterior to boundary
• secreted, activates own expression (autocrine)
  and that of engrailed

Positive feedback loop that ensures a cellular
memory of the position of the boundary --local
paracrine signaling
Ligands receptors
hh
Patched
wg
Frizzled
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cellular memory

• boundaries form at time of cellularization, so
  need cell-cell signaling pathways
• three mechanisms ensure that engrailed turns on
  and stays on in the posterior compartment
• wg/hh ve feedback loop (paracrine signaling)
• engrailed autoactivation (cell-autonomous)
• stabilization of chromatin states
  (cell-autonomous more later)

38
compartment boundaries

• engrailed (etc) accounts for the difference
  between anterior and posterior, but does not
  explain
• why the boundary is straight
• why cells do not mix
• differences in cell adhesion between A and P
  compartments could lead to sorting-out, with
  straight line being energetically favorable
• the cell adhesion molecules involved are still
  unknown!

39
how do we get from parasegment to segment?

• parasegments are developmental units, but
  segments are the functional units (wing, leg)
• the parasegment boundary may act as a pattern
  organizer for the segment around it
• cells at boundary are morphogen sources
• old evidence from cut-n-paste studies on other
  (bigger) insects (Galleria, Oncopeltus--see end
  of Chapter 6)
• in Drosophila best evidence from studies of
  imaginal discs -- to be discussed later

40
Why parasegments?

• embryo sets up self-maintaining boundary--intact
  despite 1000-fold growth of tissue from embryo to
  adult
• cell sorting ensures straight line of
  cells--spatially reliable morphogen source (Wg
  or Hh?)
• pattern each segment from center, not edge

41
is segmentation conserved?

• some gap and pair-rule genes are conserved
• engrailed stripes highly conserved
• but fundamental difference most embryos are
  cellularized, not a syncytium

expression of engrailed in spider embryos
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The segment polarity machine is robust

• model can generate periodic pattern from variety
  of initial conditions
• maybe it can be set in motion by spatial cues
  (Drosophila) or temporal cues (if segments formed
  progressively)

(von Dassow et al., 2000)
43
review AP patterning

• Axis specification
• gradients
• Segmentation
• regulatory hierarchy of genes
• form boundaries of parasegments, then organize
  segments