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

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.

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. DevBio9e-Fig-06-01-0.jpg

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.

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

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).

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.

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. DevBio9e-Fig-06-03-0.jpg

Nuclear and cell division in Drosophila A = embryo in the syncytial stage B = cellular blastoderm DevBio9e-Fig-06-02-0.jpg

Fruit Fly Developmental Stages & Life Cycle

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

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.

Gastrulation in Drosophila DevBio9e-Fig-06-04-0.jpg

Gastrulation in Drosophila (Part 4) This is the 1st instar larvae that is readily visible in a fruit fly culture. DevBio9e-Fig-06-04-4R.jpg

1st Instar

Schematic representation of gastrulation in Drosophila DevBio9e-Fig-06-05-0.jpg (Late in B & Early into C): Start of gastrulation Left = what will become ventral body surface Right = dorsal surface

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 DevBio9e-Fig-06-06-0.jpg

(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.

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).)

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. DevBio9e-Fig-06-07-1R.jpg

The anterior-posterior axis is specified during oogenesis (Part 2) DevBio9e-Fig-06-07-2R.jpg

The anterior-posterior axis is specified during oogenesis (Part 3) DevBio9e-Fig-06-07-3R.jpg

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. DevBio9e-Fig-06-09-0.jpg

Generating dorsal-ventral polarity in Drosophila DevBio9e-Fig-06-10-0.jpg

(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. DevBio9e-Fig-06-11-0.jpg

Gastrulation in Drosophila DevBio9e-Fig-06-13-0.jpg

Dorsal-ventral patterning in Drosophila DevBio9e-Fig-06-15-0.jpg

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. DevBio9e-Fig-06-16-0.jpg

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. DevBio9e-Fig-06-18-0.jpg

DevBio9e-Table-06-01-1R.jpg

Syncytial specification in Drosophila Syncytial – a multinucleated mass of cytoplasm that is not separated into individual cells. DevBio9e-Fig-06-19-0.jpg

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. DevBio9e-Fig-06-20-0.jpg

Bicoid mRNA and protein gradients shown by in situ hybridization and confocal microscopy DevBio9e-Fig-06-21-0.jpg

***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. DevBio9e-Fig-06-22-0.jpg

Model of anterior-posterior pattern generation by Drosophila maternal effect genes DevBio9e-Fig-06-23-0.jpg

Bicoid protein gradient in the early Drosophila embryo DevBio9e-Fig-06-24-0.jpg

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 DevBio9e-Fig-06-26-0.jpg

Homeotic gene expression in Drosophila Homeotic genes cause the development of specific structures in plants and animals. DevBio9e-Fig-06-35-0.jpg

A four-winged fruit fly constructed by putting together three mutations in cis-regulators of the Ultrabithorax gene DevBio9e-Fig-06-36-0.jpg

End.