1. Axis Specification I

2. The Maternal-Effect Mutants
Drawing is in CNV commentary in supplement to Cell Reviews issue Jan. 2004
(also found the D/V Group)
Nusslein-Volhard, 2004

3. A-P polarity set up in egg chamber
bicoid mRNA oskar mRNA
protein + nanos RNA
microtubule-based

4. The Maternal A/P Information
BCD is a transcription factor and a translation factor (represses Caudal translation)
NANOS is a translation factor (represses Hunchback translation)
The Terminal System
BCD
NANOS

5. The Zygotic Genes
Nusslein-Volhard and Wieschaus, Nature Wieschaus Nobel Lecture, 1995

6. Creating Complex Patterns From Simple Ones
maternal
zygotic
BCD Gradient
GAP Genes
HB = Red, KR = Green
Pair Rule Gene
(fushi tarazu)
Segment Polarity
(engrailed)

7. Complex Patterns From Simple Ones: Gap Genes
BCD
Giant
Kruppel

8. 8 Pair-rule genes x 7 stripes =
Creating Complex Patterns From Simple Ones
3 Maternal “Systems” for A/P
maternal
zygotic
≈11 Gap Gene Domains
8 Pair-rule genes x 7 stripes =
56 domains
eve
ftz

9. Generating Precise Positional Information Along Axes
Dave Kosman

10. Understanding Transcriptional Control of Patterning: The Story of eve
Levine and colleagues
eve is a pair rule gene--expressed in 7 “stripes”
How is this pattern created?
Step 1: “Promoter bashing” to identify relevant enhancers
eve
ftz
eve Stripe 2 enhancer--lacZ
S. Small
eve Stripe 3/7 enhancer--lacZ

11. Studying Transcriptional Control of Development
Step 3: Use biochemistry to identify direct regulators
Eve
Step 1: “Promoter bashing” to identify relevant enhancers
Step 2: Use expression patterns and genetics to identify candidate regulators
eve stripe 2 Gt
Step 4: In vivo proof: mutating cis binding sites should cause same change in expression as mutating trans regulators Kr
Small, Blair and Levine EMBO 1992

12. Eisen Lab Sepsidae (scavenger flies) ≈100 mya PMID: 18584029
Study family sepsidae to find flies far enough away from Drosophila for good sequence divergence, but close enough to still have similar pattern of development
Black scavenger flies--must be grown on cow dung (Mike is a little crazy)
Had to work out conditions to fix and stain Sepsidae embryos
Had to clone out different genes of interest
Had to sequence eve locus from these different species
≈100 mya

13. Can find homology in enhancers within species in the same family
Difficult to find homology between families (eve obviously conserved)
Eisen Lab
PMID:

15. Eisen Lab But, the Sepsidae enhancers work in Drosophila
Therefore even dramatically rearranged enhancers can have same function
Makes it difficult to understand what is required about an enhancer
Makes it VERY difficult to predict enhancers, either within a species or between species
Eisen Lab
PMID:

17. Positional Information in the Embryo Controls:
Segment Identity
(Making Segments Different From One Another)
Segmentation and
Pattern Within a Segment

18. Segment Polarity Genes
-wg and hh Signaling Pathways
-Establish 14 Segmental Boundaries
-Act as morphogens to pattern within each segment Hh Wg

19. Positional Information in the Embryo Controls:
Segment Identity
(Making Segments Different From One Another)
Segmentation and
Pattern Within a Segment

20. Fun with Flies!
Homoeosis (Bateson 1894): alteration of one organ of a segmental or homologous series from its own characteristic form to that of another member of the series “homoeotic mutants”
Ultrabithorax
Antenae to Leg
Leg to Antenae
Proboscis to Leg
Haltere to Wing
Wing to Haltere
Genitalia to Leg
Ed Lewis

21. Homeotic (Hox) genes are expressed differentially along the A/P Axis
Specify segment identity

22. Drosophila
Mouse

23. Drosophila
Hypothetical
common ancestor
Amphioxus
Mouse

24. Regulation of Segment Identity by a “Hox Code”
Wild Type
T2 T3
Antp ON Antp OFF
Ubx OFF Ubx ON
Wing Haltere
Ubx mutant
T2 T3
Antp ON Antp ON
Ubx OFF Ubx OFF
Wing Wing
Ultrabithorax
Wing
Haltere
UBX Expression

25. Mammals Are Segmented Too
Cervical
Thoracic
Lumbar
Sacral

26. Hox Genes Specify A/P Identity in Vertebrates
5/6 mutant
Triple homozygote
A/a C/c D/c X
1/64 pups = aaccdd
Redundancy has plagued hox gene knockouts
-must have functional specificity to be conserved, but individual knockouts don’t give stunning phenotypes
Knock out all 6 alleles of Hox10
-genetic tour de force
-3/6 mutant: viable and fertile
-4/6 mutant: infertile
-5/6 mutant: one extra rib
-6/6 mutant: ribs on all lumbar and sacral vertebrae!!!!!!
Wellik and Capecchi, 2003

27. Patterning the Dorsal-Ventral Axis
Maternal-Effect Mutants: The D/V Group D wt A P V
Dorsalized
Ventralized

28. D/V Patterning Establishes the Mesodermal Identity Determined by twist and snail
A. Courey
Toll pathway: on on ventral side

29. It is not birth, marriage or death, but gastrulation, which is truly the most important time in your life.
              - Lewis Wolpert (1986)
Gastrulation

30. Drosophila gastrulation
The mesoderm and endoderm invaginate separately
(a bit unusual)
Ventral View

31. ? Cell Adhesion and Cell Sorting Cell Identity Morphogenesis
Cell Biology
Cell Division/Death Cell Adhesion Cell Movement Cell Shape

32. The Cell Biology of Fly Mesoderm Gastrulation
Epithelial-mesenchymal transition (EMT)
Apical constriction
Invagination
Migration/spreading

33. folded gastrulation (ligand)
folded gastrulation and concertina Effect Apical Constriction
twist and snail
folded gastrulation (ligand)
Apical constriction
fog expression in mesoderm is dependent on twist and snail
GPCR?
Concertina(G alpha)
Fog expression in mesoderm downstream of twist/snail
Both only partially blocked (delayed) invagination
Double mutants should be same as single if in same pathway
Hsfog can induce ectopic constrictions--this is blocked by cta
Activated cta can also induce ectopic constrictions, this is NOT blocked by fog-
Therefore cta is downstream of fog
Hypothesize a GPCR in between
A little genetics:
Mutations iin RhoGEF have more severe phenotype
Suggest second pathway activating RhoGEF
Recently found--T48 transmembrane protein
T48 expression also controlled by twi/sna
IF T48 and fog/cta are really parallel pathways, expect double mutant to be more severe and look like RhoGEF
This is what they found (in contrast to predicted result if in same pathway--no additive effect)
RhoGEF

34. folded gastrulation (ligand)
folded gastrulation and concertina Affect Gastrulation
twist and snail
folded gastrulation (ligand)
GPCR?
T48
Concertina(G alpha)
fog
RhoGEF
Rho Kinase
myosin
Apical constriction
Myosin relocalizes from basal to apical at the time of apical constriction
Fog expression in mesoderm downstream of twist/snail
Both only partially blocked (delayed) invagination
Double mutants should be same as single if in same pathway
Hsfog can induce ectopic constrictions--this is blocked by cta
Activated cta can also induce ectopic constrictions, this is NOT blocked by fog-
Therefore cta is downstream of fog
Hypothesize a GPCR in between
A little genetics:
Mutations iin RhoGEF have more severe phenotype
Suggest second pathway activating RhoGEF
Recently found--T48 transmembrane protein
T48 expression also controlled by twi/sna
IF T48 and fog/cta are really parallel pathways, expect double mutant to be more severe and look like RhoGEF
This is what they found (in contrast to predicted result if in same pathway--no additive effect)
T48 is membrane protein that physically interacts with RhoGef

35. DE-cadherin May Regulate EMT in the Drosophila Mesoderm
Epithelial-mesenchymal transition (EMT)
snail
DE-cadherin
EMT

36. FGF Signaling Promotes Mesoderm Migration
Twist and snail promote FGFR expression in mesoderm and repress FGF expression in mesoderm
(FGFR)

37. How Cell Identity Controls Morphogenesis of Gastrulation
Cell Identity (twist, snail)
fog, T48
FGF-R
Cell Adhesion
E-Cadherin?
Migration
Epithelial-mesenchymal transition (EMT)
Apical constriction