1. The Genetics of Axis Specification in Drosophila
BIOL 370 – Developmental Biology
Topic #8
The Genetics of Axis Specification in Drosophila
Lange

2. While we know a large amount about fruit fly genetics due to decades of research beginning most notably with Thomas Hunt Morgan (Nobel Prize in 1933) and his students, fruit fly development is not as well understood in some ways, due to its complexity.

3. Laser confocal micrographs of stained chromatin showing superficial cleavage in a Drosophila embryo
We see here syncytial eggs. Synctyial cells are defined as having a multinucleated mass of cytoplasm that is not separated into individual cells.
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4. From what we do know, however, especially interesting for Drosophila development is:
CELL MEMBRANES do not form until after the 13th nuclear division.
Prior to the 13th nuclear division, the nuclei share the same “common” cytoplasm
This allows information from each nuclei to diffuse through the entire embryo until that time. (Potential value?)
Cell specification occurs by the interactions of the components within a SINGLE multinucleated cell.

5. Fertilization in Drosphila:
Sperm enters an ACTIVATED EGG.
Activation of the egg occurs during ovulation
When the female oviposits (lays) her eggs, the pressure of going through the ovipositor opens calcium channels in the membrane of the oocyte.
The open calcium channels allows calcium to flood into the oocyte.
Due to this influx of calcium, the nucleus of the oocyte initiates meiotic processes associated with nuclear division

6. For the male in fertilization, there is only one site on the egg where sperm can enter…
The micropyle:
This structure is actually a channel/tunnel in the chorion of the egg (chorion is the name given to the shell of the egg).

7. Drosophila sperm have extremely long tails. In D
Drosophila sperm have extremely long tails. In D. melanogaster, the length of a single sperm can be up to 1.8mm in length.
This is almost as long as the adult fly, is longer than the egg, and is about 300X longer than a human sperm.

8. Formation of the cellular blastoderm in Drosophila
From the syncytial blastoderm at roughly division 10, microtubules begin to surround the nuclei creating a nucleus/cytoplasm/microtubular “island” called an ENERGID.
Following division 13, the egg cell membrane begins to fold inward, creating the cellular blastoderm.
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9. Nuclear and cell division in Drosophila
A = embryo in the syncytial stage
B = cellular blastoderm
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10. Fruit Fly Developmental Stages & Life Cycle

11. Gastrulation in Drosophila:
While they appear vastly different, the general body plan of the multicellular embryo, the larva, and the adult are the same.
Head region
Multilple (11) segmental units
Tail region

13. Each of these 11 segments has a unique identity as well
Each of these 11 segments has a unique identity as well. For example in the first three thoracic segments:
1 = legs
2 = legs and wings
3 = legs and halteres
Halteres are small knobbed structures modified from the hind wings in some two-winged insects (like Drosophila). They are moved rapidly in a countercurrent pattern to the wins and function as gyroscopes. They help with balance in flight and informing the insect about rotation of the body during flight.

14. Gastrulation in Drosophila
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15. Gastrulation in Drosophila (Part 4)
This is the 1st instar larvae that is readily visible in a fruit fly culture.
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16. 1st Instar

17. Schematic representation of gastrulation in Drosophila
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(Late in B & Early into C):
Start of gastrulation
Left = what will become ventral body surface
Right = dorsal surface

18. H segment = head segment T segments = thorax segments
Comparison of larval (left) and adult (right) segmentation in Drosophila
H segment = head segment
T segments = thorax segments
A segments = abdominal segments
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19. (A) Early cleavage (15 minutes–1. 5 hours)
(A) Early cleavage (15 minutes–1.5 hours). Nuclei cleave in the central region.
(B) Migration of cleavage nuclei (1.5 hours). Nuclei migrate to the periphery.
(C) Formation of syncytial blastoderm (2 hours). Pole cells form posteriorly.
(D) Cellular blastoderm (2.5 hours). Cell membranes form between the nuclei.
(E) Early gastrulation (3.5 hours). The ventral furrow forms. Thickening of the posterior plate below the pole cells.
(F) Midgut invagination (3.5–4 hours). The midgut invagination can be seen ventrally, as can the cephalic furrow.
(G) Germ band extension (4–5 hours). Invagination of the hindgut can be seen dorsally.
(H) Stomodeal invagination (5–7 hours). Invagination of the stomodeum can be seen ventrally.
(I) Shortening of the germ band (9–10 hours). Foregut and hindgut invaginations are deep.
(J) Shortened embryo (10–11 hours). Hindgut is now fully posterior.
(K) Dorsal closure (13–15 hours). The ectoderm closes dorsally. The midgut broadens. The head involutes.
(L) Condensation of ventral nervous system (15 hours–hatching). The gut regions are joined. The nervous system forms ventrally.

20. The Value of Cell Polarity:
Organ development is a carefully scripted process of cellular proliferation, differentiation, and apoptosis.
Persistently delineating cell polarity by identifying the apical (top of the cell) and basolateral (bottom and sides of the cell) surfaces is crucial for the proper development of organs.
Defects in this system can result in severe developmental abnormalities and embryo death.
Several of the key proteins defining cell polarity have been identified, but the role these proteins play during development is still often unclear.
(The above based upon information from G. Brennan (accessed 04/15/13).)

21. The anterior-posterior axis is specified during oogenesis (Part 1)
Gurken proteins (green spheres (aka a “pickle”) when produced by the oocyte will bind with the Torpedo receptors in the terminal follicle cells driving what will become the posterior axis.
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22. The anterior-posterior axis is specified during oogenesis (Part 2)
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23. The anterior-posterior axis is specified during oogenesis (Part 3)
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24. Germline chimeras made by interchanging pole cells between wild-type embryos and embryos from mothers homozygous for a mutation of the torpedo gene
The exchange of the pole cells affects further differentiation of the embryos.
The follicle cells that were made deficient for torpedo guided abnormal development in genetically normal embryos.
In mutant strains, follicle cells were made “wild” for torpedo guided NORMAL development in genetically abnormal embryos.
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25. Generating dorsal-ventral polarity in Drosophila
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26. (A) shows typical development of this mutation in the larval form
Effect of mutations affecting distribution of the Dorsal protein, as seen in the exoskeleton patterns of larvae
These images represent another mutation this time guiding development of only dorsal cells.
(A) shows typical development of this mutation in the larval form
(B) shows how normal appearing development can be induced via injection of wild-type mRNA.
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27. Gastrulation in Drosophila
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28. Dorsal-ventral patterning in Drosophila
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29. Left-right axis formation in Drosophila involves the microfilament cytoskeleton
The Drosophila also has a distinct right/left division and is not simply a mirror image bilaterally. Areas that most readily show this asymmetry include the hindgut (HG), and the male (MG) and female (FG) gonads.
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30. Normal and irradiated embryos of the midge Smittia
Effects of radiation (UV light) on the early development of the midge can be profound if they alter ventral/dorsal polarity. Here the bottom (irradiated) midge displays NO HEAD and two abdominal/tail regions.
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31. DevBio9e-Table R.jpg

32. Syncytial specification in Drosophila
Syncytial – a multinucleated mass of cytoplasm that is not separated into individual cells.
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33. Schematic representation of experiments demonstrating that the bicoid gene encodes the morphogen responsible for head structures in Drosophila
In this experiment by Driever et. al. (1990), bicoid mRNA from wild type Drosophila are injected into mutant strains or abnormal locations in other wild strains. Note the results in terms of head development.
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34. Bicoid mRNA and protein gradients shown by in situ hybridization and confocal microscopy
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35. ***Note that the gradient is complementary to that of bicoid.
Caudal protein gradient in the syncytial blastoderm of a wild-type Drosophila embryo
***Note that the gradient is complementary to that of bicoid.
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36. Model of anterior-posterior pattern generation by Drosophila maternal effect genes
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37. Bicoid protein gradient in the early Drosophila embryo
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38. Three types of segmentation gene mutations
In normal, unmutated Drosophila, each segment produces bristles called denticles in a band arranged on the side of the segment closer to the head.
Gap Segmentation Mutation where the active gene differentiates the whole thorax region uniformly, and the mutant form lacks these segments.
Pair-rule mutation where alternate segments do not develop normally in the mutant form.
Segment Polairty mutation where the portion of the posterior segment does not develop in the mutant
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39. Homeotic gene expression in Drosophila
Homeotic genes cause the development of specific structures in plants and animals.
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40. A four-winged fruit fly constructed by putting together three mutations in cis-regulators of the Ultrabithorax gene
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41. End.