Drosophila Body Plan (part 2): Segmentation

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Drosophila Body Plan (part 2) Segmentation
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• Segmentation is the most obvious feature of
  Drosophila larvae
• Each segment has its own identity
• Segments are derived from parasegments (the
  first units to form)

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• 14 parasegments appear after gastrulation
• Delimited by temporary grooves
• Initially similar, but acquire unique identities
• Offset from segments a segment is made up of
  the posterior half of one parasegment and the
  anterior half of the next segment
• Anterior parasegments fuse to form the head
• Defined by expression of pair-rule genes

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• Pair-Rule gene expression is determined by gap
  genes (non-repeating pattern leads to repeating
  stripes of pair-rule activity)
• Pair-Rule genes are each expressed in 7
  transverse stripes in alternate
• parasegments (every other one)
• Some define odd numbered parasegments (even
  skipped), while some define even numbered ones
  (fushi tarazu)
• Mutations affect alternate parasegments
• Pattern is present before cellularization, which
  occurs soon after pair-rule gene expression

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anterior
posterior
even-skipped (green) and fushi tarazu (red)
expression patterns
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• Each stripe is only a few cells wide
• Some define parasegment boundaries
• Pattern appears gradually
• e.g. even skipped expression begins at low
  levels in all nuclei, narrows as other stripes
  develop, initially fuzzy, becomes more defined
• Each stripe is independently specified

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Even skipped specification

• Dependent on bicoid and 3 gap genes (hunchback,
  Krüppel, and giant)
• bicoid and hunchback protiens activate even
  skipped
• Krüppel and giant proteins repress
• even skipped
• Anterior boundary defined by giant
• Posterior boundary defined by Krüppel

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• For independent localization, each stripe must
  respond to different combinations and
  concentrations of transcription factors
• Requires complex control regions with multiple
  binding sites for each factor
• The even skipped regulatory region has several
  separate regions that control each stripes
  localization (shown using lacZ reporter gene
  discussed in Box 5A)
• Isolated regulatory regions contain 500bp
• Each determines expression of a single stripe

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(No Transcript)
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• Each regulatory region has binding sites for
  different transcription factors, some activate,
  others repress
• Gap genes regulate pair-rule expression in each
  parasegment
• Some are not directly regulated by gap genes,
  but depend on expression of other pair- rule
  genes (e.g. fushi tarazu)

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• Pair-Rule genes encode transcription factors
• These set up the final segments in the embryo
• Pair-rule gene expression is temporary and
  cellularization is occurring
• How do parasegment positions become fixed and
  how are segment boundaries formed?

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• Segment Polarity Genes
• Not all transcription factors like gap and
  pair-rule
• Diverse, unrelated, and work through different
  mechanisms
• Called segment polarity genes because mutations
  affect anterior-posterior polarity of segments
  (mirror-image or tandem duplication)
• Activated by pair-rule genes
• Each expressed in 14 stripes corresponding to the
  parasegments
• Act on cells, not syncytium

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• engrailed
• Transcription factor
• Expressed in anterior of each parasegment
• Delimits a boundary of cell lineage restriction
• Selector gene confers regional identity
  through control of other genes and act for a
  longer period (engrailed expressed throughout
  life)

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• engrailed activity begins at cellularization
• 14 stripes at anterior of each parasegment
• Initially expressed in one line of cells in each
  parasegment
• Result of combinations of pair-rule
• transcription factors
• Evidence fushi tarazu mutations
• lead to engrailed expression
• only in odd numbered
• parasegments (missing where
• fushi tarazu is expressed)

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• Anterior margin of engrailed expression is a
  boundary of cell lineage restriction
• Cells in a parasegment never move into the
  adjacent parasegment
• Suggests common genetic control (controlling
  development and preventing mixing)
• Compartments - detected by labelling a single
  cell to identify all descendents at a later
  stage
• Cell lineage studies show that all descendents
  are restricted to the specific parasegment
• Forms border between anterior and posterior
  portions of the final segments (defined by
  engrailed)

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• Minute technique can be used to show common
  genetic control of cells in a compartment in
  adult wing development
• (Box 5B)
• Normal wing compartment is constructed of all
  descendents of a founding cell
• engrailed mutants descendents of marked cell
  are not limited to one part of the segment

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• Each segment has a well-defined A-P pattern
  visible on ventral abdomen (denticles)
• Located on anterior region of each segment only
• Rows of denticles create pattern that reflects
  A-P polarity gradient
• Depends on maintenance of parasegment boundary
  (restricted segment polarity genes)
• Mutations in segment polarity genes alter pattern
• e.g. wingless and hedgehog whole abdomen has
  denticles, but in mirror-image pattern
• Anterior pattern is duplicated (in reverse),
  posterior pattern is lost

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• Denticle pattern is created by segment polarity
  genes, intercellular signalling
• hedgehog and wingless encode secreted signalling
  proteins similar to vertebrate sonic hedgehog
  and Wnt
• engrailed and hedgehog are expressed at the
  anterior margin of the parasegment boundary
• wingless is expressed at the posterior margin
• patched is expressed in cells where engrailed
  and hedgehog are not expressed

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• hedgehog encodes secreted protein that maintains
  wingless in the adjacent cell on the other side
  of the boundary
• wingless encodes a secreted glycoprotein that
  maintains engrailed and hedgehog
• Other segment polarity genes encode other
  components of these signalling pathways (e.g.
  patched encodes the receptor for the hedgehog
  protein)
• Gradients of hedgehog and
• wingless signals are set up within
• each segment boundary

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Evidence from other insects Oncopeltus and
Galleria

• Oncopeltus have hairs covering each segment
• Normally each hair points in an anterior to
  posterior direction
• Gap in the segment boundary leads to disruption
  of orientation in some individuals
• Can be explained by a morphogen gradient from
  anterior to posterior of each segment
• If the gradient gives hairs their polarity they
  will point down the gradient
• Gap in the boundary will lead to a change in the
  local gradient (opposite direction)

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• Galleria provide evidence for this gradient via
  grafting experiments
• Have 7 types of cuticle scales arranged in
  consecutive bands
• Grafting a piece of larval cuticle to a more
  anterior position leads to repatterning in that
  region of the host
• Best explained by gradient that determines
  polarity

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Other insect body plans

• Long-germ insects
• Includes Drosophila
• All segments specified at about the same time
• Blastoderm corresponds to whole future embryo
• Short-germ insects
• Includes Tribolium (flour beetle)
• Short blastoderm forms only anterior segments
• Posterior segments formed by growth after
  completion of the blastoderm and gastrulation
• Most segments formed from cellular blastoderm

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• Both long- and short-germ insects appear similar
  as mature embryos
• All share a common developmental stage
  (phylotypic stage)
• Although short-germ insects lay down much of
  their body plan later than Drosophila (after
  cellularization), the same genes are involved
• e.g. Krüppel is expressed in blastoderm stage in
  the posterior end of Tribolium (different area,
  but same body region)
• 2 pair-rule repeats posterior cap
• wingless and engrailed expression

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• Most genes have not been studied in insects other
  than Drosophila, but a few are known
• engrailed is expressed in posterior segments of
  many insects
• even skipped is expressed in grasshoppers
  (short-germ insects), but plays a different
  role in their development
• Nervous system
• Growing posterior germ band

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• Leaf Hoppers (Euscelis) have anterior-posterior
  axis determining mechanism similar to the
  bicoid gradient
• Evidence comes from 2 experiments
• Ligature tied around the fertilized egg causes a
  gap in the body plan
• Eggs have a posterior ball of symbiotic bacteria
• Ball is moved anteriorly with a microneedle,
  taking some cytoplasm along
• Ligature tied behind ball causes development of
  normal structures anterior to the ligature and
  incomplete mirror-image structures on the other
  side

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• Parasitic wasps provide an example of more
  dramatic differences
• Small eggs form a ball of cells during cleavage
• Ball falls apart forming up to 400 cell clusters
• Each cluster can develop into a separate embryo
• Not dependent on maternal information for body
  axis specification

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Homeotic Selector Genes

• Segment polarity genes are turned on in each
  segment
• Homeotic selector genes specify the identity of
  each segment
• Selector genes are a class of regulatory genes
  (control activity of other genes)
• Organized into 2 gene complexes in Drosophila
• Together they are homologous to a single
  vertebrate Hox gene complex
• Homeotic genes code for transcription factors
  that contain a homeobox sequence (180bp,
  conserved)
• Control patterning in vertebrates too, but first
  identified and best understood in Drosophila

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• 2 homeotic complexes
• Named for mutations that
• revealed existance
• Bithorax part of haltere on
• 3rd thoracic segment is
• transformed into part of a wing
• Antennapedia dominant
• mutations transform
• antenae into legs
• Homeosis is the
• transformation of one
• segment into another related one

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• Transformations occur due to the role of selector
  genes in positional identity
• Control activity of other genes in a segment
• e.g. will an imaginal disc develop as a wing, or
  a leg?

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• Bithorax controls development of parasegments
  5-14
• Antennapedia controls anterior parasegments
• Bithorax is best understood (discussed first)
• 3 genes (regulated by gap pair-rule)
• ultrabithorax (parasegment 5 onward)
• abdominal-A (parasegment 7 onward)
• abdominal-B (parasegment 10 onward)
• abdominal-B suppresses ultrabithorax (low in 14)

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• The role of genes in the bithorax complex was
  shown using classical experiments in which all
  or parts of the complex were missing
• Larvae lacking the whole bithorax complex
  parasegments 5-13 all resemble parasegment 4
  (basic pattern represents default state)
• Genes put back one at a time to deduce the role
  of each
• Ultrabithorax alone one parasegment 4, one
  parasegment 5, and eight parasegment 6
• Abdominal-A ultrabithorax parasegments 4, 5,
  6, 7, 8, and five parasegment 9
• Abdominal-B added normal development
  (expression highest in parasegment 14)
• Differences between segments reflect spatial and
  temporal pattern of homeotic gene expression

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• Bithorax genes act in a combinatorial manner to
  specify parasegments (seen by removing genes
  one at a time)
• Ultrabithorax absent parasegments 5 and 6
  converted to 4, effect on cuticle pattern in
  7-14 (characterisctics of thorax in abdomen)
• Expression of combinations not normally present
  leads to these abnormalities (nonsense
  combinations, abdominal-A without ultrabithorax)

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• Gap and pair-rule genes control the pattern of
  homeotic gene expression, but their proteins
  disappear after about 4 hours
• Other genes are needed to maintain continued
  expression of homeotic genes
• 2 groups of genes are involved
• Polycomb maintains transcritional repression in
  cells where homeotic genes are off
• Trithorax maintains expression in cells where
  homeotic genes are on

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• Antennapedia complex has five homeobox genes
• Work on same principle
• Deformed mutations affect parasegments 0 and 1
• Sex combs reduced - mutations affect parasegments
  2 and 3
• Antennapedia - mutations affect parasegments 4
  and 5
• Labial and proboscidea mouth parts??

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• In both homeotic gene comlpexes the genes occur
  in the same spatial and temporal order (3-5)
  in the complex as their expression (A P) in
  the embryo
• Same pattern occurs in vertebrate Hox genes
• Highly conserved co-linearity probably related to
  the mechanisms controlling expression

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• Complex control of bithorax complex is shown in
  Drosophila engineered to express ultrabithorax
  in all segments
• Ultrabithorax coding sequence is linked to a
  heat- shock promoter (activated at 290C)
• Introduced using a P element
• Heat-shock induces high levels of expression in
  all cells
• No effect in most posterior segments that
  normally produce ultrabithorax
• Parasegment 5 transformed into 6
• Anterior parasegments also transformed into 6
• Parasegment 13 not affected (normally suppressed,
  somehow inactivated)
• Role of downstream genes is not well known

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Homeotic gene expression in visceral mesoderm
controls structure of the adjacent gut

• Engrailed and bithorax complex genes are
  expressed in the somatic and visceral mesoderm
• Somatic mesoderm gives rise to main body muscles
• Visceral mesoderm pattern induces gut endoderm
• Developing midgut has 3 constrictions
• Second constriction is in parasegment 7
• Most homeotic genes are not expressed in endoderm
• Specificity induced by expression in surrounding
  somatic mesoderm
• Visceral mesoderm pattern of bithorax expression
  is different than ectoderm or somatic mesoderm

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• Ultrabithorax is only expressed in the
  parasegment of visceral mesoderm adjacent to the
  second gut constriction (parasegment 7)
• If absent, constriction does not develop and gut
  is abnormal
• Ultrabithorax is not expressed in the endoderm,
  but acts through decapentaplegic and labial
• Dpp and labial are expressed in the area of
  constriction, but hardly expressed in absence of
  ultrabithorax (necessary to activate them)

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• Ultrabithorax protein activates decapentaplegic
  in visceral mesoderm
• Decapentaplegic protein diffuses into adjacent
  endoderm
• Stimulates signalling pathway that activates
  labial
• Labial is involved in gut morphogenesis
• Transfer of positional information from one germ
  layer to another is similar to vertebrate
  nervous system induction