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 byKrüppel

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 stripe’s localization (shown using lacZ reporter gene – discussed in Box 5A) • Isolated regulatory regions contain ~500bp • Each determines expression of a single stripe

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. posterior anterior 5´ 3´ • 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. posterior anterior 5´ 3´ • 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. posterior anterior 5´ 3´ • 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