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Drosophila Body Plan (part 2): Segmentation

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Title: Drosophila Body Plan (part 2): Segmentation


1
Drosophila Body Plan (part 2) Segmentation
2
  • 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)

3
  • 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

4
  • 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

5
anterior
posterior
even-skipped (green) and fushi tarazu (red)
expression patterns
6
  • 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

7
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

8
  • 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

9
(No Transcript)
10
  • 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)

11
  • 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?

12
  • 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

13
  • 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)

14
  • 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)

15
  • 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)

16
  • 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

17
  • 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

18
  • 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

19
  • 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

20
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)

21
  • 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

22
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

23
  • 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

24
  • 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

25
  • 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

26
  • 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

27
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

28
  • 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

29
  • 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?

30
  • 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)

31
  • 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

32
  • 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)

33
  • 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

34
  • 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??

35
  • 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

36
  • 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

37
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

38
  • 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)

39
  • 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
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