Drosophila Oogenesis and Fertilization

Drosophila Development

• Textbook: Chapter 10, pages 303-310, 312-330
• Chapter Overview: Drosophila fertilization, cleavage, anterior-posterior axis establishment, and dorsal-ventral axis establishment

Drosophila

• Classification: Phylum - arthropod, Class - insecta, Protostome
• Model Organism: Thomas Hunt Morgan introduced the fruit fly as a model organism in the early 1900s.
• Genome/Mutations: Complete genome sequence known, easy mutant generation

Drosophila Lifecycle

• Stages: Embryo, larva, pupa, adult
• Embryogenesis: Syncytial cleavage until 13th cell division, cellularization, gastrulation/segmentation, organogenesis
• Larva: Embryo hatches as a larva, 3 instars (separate by molts)
• Pupa: Larvae pupate at end of 3rd instar, remodeling for adult structures (metamorphosis)
• Adult: 9-10 days after fertilization, adult fly emerges

Drosophila Oogenesis

• Ovaries: Pair, 15-20 ovarioles/egg tubes
• Egg Chambers/Arrangement: Arranged linearly, least mature near germarium, mature furthest
• Germarium: Apical structure, stem cell location
• Egg Chambers: Clusters of cells surrounded by follicular cell, Each chamber produces one mature egg
• Oogenesis Stages: Stem cell divides incompletely (no cytoplasmic cleavage) 4 times, generating 16 cells connected by cytoplasmic bridges. 15 of these cells form nurse cells; 1 cell becomes the oocyte. Oocyte matures as the egg chamber moves away from the germarium.
• Nurse Cells: Synthesize maternal proteins and mRNAs stored in oocyte, undergo apoptosis at end of oogenesis.
• Mature Oocyte: Released into oviduct, surrounded by follicular cells, paused at metaphase I until ovulation.

Drosophila Fertilization

• Different Activation: Sperm enters an egg already activated.
• Ca2+ channels: Open when oocytes enter the oviduct due to mechanical pressure. Ca2+ initiates meiosis and translation of maternal mRNAs.
• Single Entry Site: Only one site (micropyle) for sperm entry, prevents polyspermy (multiple sperm). No cortical granules.

Drosophila Cleavage

• Superficial Meroblastic: Nuclei divide without cytokinesis (karyokinesis) until cycle 13.
• Syncytial Stage: Nuclei divide in the center until cycle 8.
• Posterior Pole Cells: ~5 nuclei reach posterior pole by cycle 9, form pole cells and create membranes that turn into gametes.
• Cycle 10: Nuclei migrate to periphery, mitosis continues at a slower rate.
• Energids: Each nucleus + actin filaments and cytoplasmic islands
• Division 13: Cell membrane folds inward, creating individual cells. The cellular blastoderm forms after 3 hrs. This process can be blocked by inhibiting microtubules, and the cellular blastoderm forms 3 hrs. post fertilization.
• Mid-blastula Transition: Cycle 14, asynchronous divisions; maternal proteins/mRNAs breakdown, zygotic transcription begins

Gastrulation

• Rapid Cell Movement: Begins shortly after the mid-blastula transition.
• Ventral Furrow: Prospective mesoderm cells (~1,000) fold inward to create ventral furrow, initiating gastrulation.
• Furrow Closure: Furrow closes at the top to form a closed ventral tube.
• Ectoderm/Cephalic Furrow: Ectoderm bends to form the cephalic furrow
• Pole Cells: Internalize at the posterior end.

Gastrulation Cont'd

• Germ Band: Collection of cells along the ventral midline forming the trunk of the embryo, extends posteriorly and wraps around the dorsal surface.
• Posterior Embryo Structures: More posterior structures are located behind the head during germ band extension.

Drosophila Body Plan

• Repeating Units: Embryo, larva, and adult have the same body plan (head, tail, repeating segments).
• Segments: Thorax (3 segments), abdomen (8 segments)
• Identity: Each segment has its own identity.

Genetic Mechanisms Patterning the Drosophila Body

• Forward Genetic Approach: Identifies genes by introducing mutations to examine mutant phenotypes and identifying associated genes
• Nobel Prize (1995): Work by Edward Lewis, Christiane Nüsslein-Volhard, and Eric Wieschaus

Hierarchy of Genes

• Maternal Effect Genes: Code for transcription factors, translational regulators (morphogens)
• Gap Genes: The first zygotic genes to be expressed, regulated by maternal effect genes.
• Pair-rule genes: Regulated by gap genes, expression at segment boundaries.
• Segment Polarity Genes: Regulated by pair-rule genes, expression in specific regions of each segment.
• Homeotic Selector Genes: Regulated by other genes; specify segment identity.

Maternal Gradients

• AP Axis: Determined by two maternal mRNAs: bicoid and nanos,
• Bicoid: Specifies anterior structures (head, mouth, thorax)
• Nanos: Specifies posterior structures
• Tethered mRNAs: Bicoid at anterior, nanos at posterior; bound via protein to 3’UTR.

Hunchback and Caudal

• Maternal mRNAs: Hunchback and caudal are other critical maternal mRNAs for anterior-posterior patterning.
• Even Distribution: Distributed evenly across the oocyte.
• Post-fertilization Translation: Translated after fertilization to form gradients.
• High Levels: High levels of hunchback at anterior, high levels of caudal at posterior.
• Translation Regulation: Bicoid and Nanos regulate translation of specific mRNAs

How Bicoid Produces Anterior Structures

• Repression of Caudal Translation: Bicoid represses translation of caudal mRNA, creating a region without caudal or inducing structures to the posterior.
• Transcription Factor Action: Bicoid acts as a transcription factor, activating the expression of target genes in the anterior embryo.
• Maternal and Zygotic Sources: Hunchback protein in early embryos originate from maternal and zygotic mRNAs.

Terminal Gene Group

• Acron and Telson: These structures (acron-head, telson-tail) form terminal portion, generated via maternal mRNAs tailless and huckebein proteins spread through cytoplasm, affecting terminal region development. Bicoid presence affects acron formation, while its lack leads to telson formation.
• Maternal mRNA Diffusion: Tailless and huckebein proteins diffuse, influencing terminal region development
• • Bicoid Influence: Bicoid presence leads to acron formation, its absence to telson formation.

Summary of AP Axis Formation

• Three Gene Sets: AP axis is defined by three sets of genes specifying anterior, posterior, and terminal regions.
• Key Gene Groups: Bicoid/Hunchback, Nanos and Caudal, and Tailless/Huckebein

Segmentation Genes

• Maternal Effect (ME) Genes: Activate zygotic segmentation genes.
• Gap Genes: Lack large sections of segmented body.
• Pair-rule Genes: Lack every other segment.
• Segment Polarity Genes: Defects in, duplications and polarity reversals in every segment.

Practice Problems

• Various Drosophila development practice problem questions to identify the roles of genetic mechanisms.