Drosophila Development Lecture Notes PDF

Lecture 14: Drosophila development Textbook Chapter 10 Pages 303 – 310, 312 – 330 Chapter overview Drosophila fertilization Drosophila cleavage Establishment of the body plan along the anterior-posterior axis Establishment of the dorsal-ventral axis Drosophila Phylum – arthrop...

Lecture 14: Drosophila development Textbook Chapter 10 Pages 303 – 310, 312 – 330 Chapter overview Drosophila fertilization Drosophila cleavage Establishment of the body plan along the anterior-posterior axis Establishment of the dorsal-ventral axis Drosophila Phylum – arthropod Class – insecta Protostome Thomas Hunt Morgan introduced the fruit fly as a model organism in the early 1900s. We know the complete genome sequence and can generate mutants easily. Life cycle of Drosophila 4 life stages: embryo, larva, pupa, adult. Embryogenesis: = Syncytial cleavage until 13th cell division Cellularization Gastrulation//segmentation Organogenesis Life cycle of Drosophila I Larva: Embryo hatches as a larva. Larvae go through 3 stages or instars, separated by molts. I Pupa: Larvae pupate at the end of the 3rd instar stage. Pupation involves a remodeling of the body and generation of adult structures like head, wings, legs, etc. (metamorphosis). A Adult: 9-10 days after the egg is fertilized, an adult fly emerges. Drosophila oogenesis Pair of ovaries consisting of 15-20 ovarioles/egg tubes. = Egg chambers are arranged linearly in the ovariole, with the egg chambers containing the least mature eggs near the = germarium, - and the most mature chambers the furthest from the germarium. Follicular cells Germarium: the most apical structure where Ovariole: stem cells are located. Egg chambers: cluster of cells surrounded by follicular cells. Each chamber will give rise to 1 mature egg. Hinnant et al. 2020. Coordinating Proliferation, Polarity, and Cell Fate in the Drosophila Female Germline. Frontiers in Cell and Developmental Biology. Drosophila oogenesis Oogenesis: A stem cell divides incompletely (without cytoplasmic cleavage) 4 times to generate 16 cells connected by cytoplasmic bridges. - 15 of these cells will become nurse cells. 1 = will become the oocyte. - The oocyte matures as the egg chamber Follicular moves away from the germarium. cells Hinnant et al. 2020. Coordinating Proliferation, Polarity, and Cell Fate in the Drosophila Female Germline. Frontiers in Cell and Developmental Biology. Drosophila oogenesis M Nurse cells synthesize maternal proteins E and mRNAs that will be stored in the oocyte. The nurse cells undergo apoptosis at the - end of oogenesis. - Follicular A-mature oocyte will be released into the cells - - oviduct to await fertilization. The oocyte is - - surrounded - by follicular cells. Drosophila oocytes are paused at metaphase I until ovulation. Hinnant et al. 2020. Coordinating Proliferation, Polarity, and Cell Fate in the Drosophila Female Germline. Frontiers in Cell and Developmental Biology. Drosophila fertilization Fertilization in Drosophila is different than what we’ve described previously. 1. The sperm enters an egg that has already been - activated. - Ca2+ channels open when oocytes enter the oviduct due to mechanical pressure. Ca2+  resumption of meiosis and translation of maternal cytoplasmic mRNAs Drosophila fertilization 2. There is only one site where the - sperm can enter the egg. - There is a tunnel in the chorion (eggshell) located at the future dorsal anterior region that allows sperm to pass one at a time, the Emicropyle. This prevents polyspermy. - =No cortical granules. Micropyle Drosophila fertilization 3. Sperm enters an egg that has already begun to specify the body Anterior-posterior axis specification in the egg chamber: - axes. - I mienstubule Drosophila Don Juan (Dj) protein: sperm fertilization specific protein 4. Sperm and egg cell membranes do - not fuse. The sperm enters the egg intact. because of the hole aka the micropole - No acrosome reaction to enter the egg. It is - hypothesized that the acrosome reaction occurs inside the egg to allow the breakdown of the plasma membrane surrounding the sperm nucleus. The sperm - tail is eventually partially degraded and is defected after larval - hatching. - - Loppin et al. The intimate genetics of Drosophila fertilization. Open Biology. 2015 Drosophila cleavage Superficial meroblastic cleavage. Until cycle 13, the nuclei divide without cytokinesis to create a syncytium (karyokinesis). Until cycle 8, the nuclei divide in the center. During cycle 9, ~5 nuclei reach the surface of the posterior pole of the embryo. They become enclosed by cell membranes and become pole cells which give rise to the gametes in the adult. Drosophila cleavage At cycle 10, the nuclei migrate to the periphery of the egg and mitosis G continues, but at a slower rate. Drosophila cleavage Energids Actin – green Microtubules - red Cross-section of a cycle Once the nuclei reach the 10 embryo periphery at cycle 10, each - nucleus becomes surrounded by microtubules - and actin filaments. Each nucleus + its associated cytoplasmic islands is called an energid. Drosophila cleavage Division 13 takes about 25 minutes to complete, mitosis is slowing down. ye After division 13, the cell membrane folds inward between each nucleus, partitioning each energid into a cell. This creates the cellular blastoderm. This process can be blocked by inhibiting microtubules. - - The cellular blastoderm in Drosophila consists of Microtubules 6000 cells and is formed 3 hours post-fertilization. The mid-blastula transition Cycle 14 is the first cycle in which cell divisions are asynchronous. Some - groups take as little as 75 minutes, others take 175 minutes. It is during this time that the maternal-to-zygotic transition takes place. Degradation - of maternal mRNAs + proteins followed by zygotic transcription. The slowdown of nuclear division, cellularization, and increase in zygotic transcription is referred to as the mid-blastula transition.  Ventral view Ventral view Gastrulation = rapid cell movement # Gastrulation begins shortly after the mid-blastula - transition. - Prospective mesoderm (~1000 cells) folds inward to create the ventral furrow. > This is the first - large scale movement that occurs - during gastrulation. The furrow closes at the top to form a closed ventral tube in the embryo. Ventral furrow closing. Mesodermal cells Ventral furrow are inside, and surface ectoderm cells beginning to form. flank the ventral midline.  Ventral view Dorsal view Gastrulation Ectoderm bends to form the cephalic furrow. Pole cells internalized at the posterior end. remembere Themar Ventral furrow closing. Mesodermal cells are inside, and surface ectoderm cells flank the ventral midline.  Cells destined to become more Gastrulation posterior larval structures are located behind the head when the germ Fullest extension of the germ band is fully extended. band. Ectodermal cells + mesodermal cells converge and form theE germ 3band. E Germ band: collection of cells along the ventral midline that includes all the cells that will form ventral dorsal the trunk of the embryo. side side The germ band extends in the posterior direction and wraps around the top (dorsal) surface of the embryo. Gastrulation Fullest extension of the germ Slightly older embryo. Germ band has band. retracted. Several morphogenic events need to occur before hatching: Segmentation appears Germ band retracts Body plan of Drosophila General 1 body plan is the same in the embryo, larva, and adult. Each stage of life has a distinct head - end, tail end, and repeating - - segmental units in between them. - Three segments – thorax Eight segments – abdomen Each segment has its own identity. How did the segments get their identity?  Genetic mechanisms patterning the Drosophila body Male flies were fed EMS (ethyl methane sulfonate) – a potent mutagen. This work by Edward Lewis, Most of the genes Christiane Nüsslein-Volhard, involved in shaping and Eric Wieschaus led them the larval and adult to winning the Nobel Prize in forms of Drosophila 1995. were identified using a forward genetic - Two No legs Missing No Genes were often named approach. posterior certain abdominal according to their mutant ends larval segments Thenotype segments phenotype. Example: wingless ↓ genotype Gene mapping + sequencing reverse genetic genotype : > - Phenotype Genetic mechanisms patterning the Drosophila body Nüsslein-Volhard and Wieschaus identified a hierarchy of genes that establish anterior- posterior polarity and divide the embryo into a specific number of segments. Genetic mechanisms patterning the Drosophila body Hierarchy: 1. Maternal effect genes (maternal mRNAs) Code for transcription factors and translational regulatory proteins that act as morphogens. 2. Gap genes The first zygotic genes to be expressed. Their expression is regulated by maternal effect genes. - - Genetic mechanisms patterning the Drosophila body Hierarchy: 3. Pair-rule genes Their expression is regulated by gap genes. - 4. Segment polarity genes Their expression is regulated by pair-rule genes. - 5. Homeotic selector genes The protein products of gap, pair-rule, and segment polarity genes interact to regulate - expression of homeotic selector genes. - Maternal gradients: anterior-posterior polarity Anterior-posterior polarity is The AP axis is determined by two established while the oocyte is in maternal mRNAs that act as - - the egg chamber. morphogens: bicoid and nanos. 15 Bicoid specifies the# anterior structures (head, mouth, thorax). I Nanos specifies theDposterior D structures. A Both mRNAs are tethered to their respective ends by proteins that bind their 3’UTR: bicoid at the anterior end and nanos at the posterior end. Maternal gradients: anterior- posterior polarity bicoid - and nanos mRNAs are transported into the egg after being synthesized by nurse cells. - bicoid and nanos are translated after fertilization and their proteins form gradients that are critical for anterior- posterior patterning. Anchoring of bicoid and nanos mRNA in the unfertilized egg - Nurse cells produce gurken mRNA which is transported into the oocyte. - 2 gurken mRNA is translated into Gurken protein. > => & Gurken binds the = Torpedo receptor on - the follicular cells  posteriorization of - the follicle cells. z This triggers a signaling cascade which - ends in the recruitment of WPar-1 protein to the posterior edge of the oocyte cytoplasm. Par-1 protein labeled in green Anchoring of bicoid and nanos mRNA in the unfertilized egg A Par-1 organizes microtubules (minus end - moves towards the anterior end, plus end - moves towards the posterior end). - - Motor proteins transport oskar mRNAs to the plus end of microtubules (posterior end). - Motor proteins then transport bicoid mRNA to the anterior pole. - 1 nanos mRNA gets trapped in the posterior pole - & after diffusion there. - - Oskar binds nanos mRNA and prevents its degradation. Experiments demonstrating that bicoid is the gene encoding the morphogen that specifies head structures. Hunchback and caudal Hunchback and caudal are two other maternal mRNAs critical for anterior-posterior patterning. hunchback and caudal mRNAs are distributed evenly across the oocyte. Hunchback and caudal hunchback and caudal are translated after - - fertilization. -- hunchback and caudal proteins form gradients across the AP axis: high - - hunchback at the anterior end and high -- caudal at the posterior end. -D How does this happen? Hunchback and caudal Bicoid and Nanos proteins are regulators of translation. Bicoid prevents translation of -caudal mRNA. -Nanos prevents - translation of -hunchback mRNA. The result of these interactions is the creation of four maternal protein gradients after fertilization. All four of these proteins act as morphogens. How does Bicoid produce anterior head structures? 1. Represses the translation of caudal which would induce posterior structure formation. 2. Acts as a transcription factor that activates the expression of target genes in the anterior part of the embryo. Anterior gap genes + zygotic hunchback. The Hunchback protein that is translated in the early embryo comes from maternal mRNA + zygotic mRNA. The terminal gene group There is a third set of maternal mRNAs whose proteins generate the unsegmented extremities of the anterior- posterior axis: the acron and the telson. The acron is the terminal portion of the head that includes the brain. The telson is the tail. The maternal mRNAs tailless and huckebein are responsible for their formation. Both mRNAs are at both poles. Tailless and Huckebein proteins diffuse into the cytoplasm from the poles. If Bicoid is present, the terminal region forms an acron. If no Bicoid is present, the terminal region forms a telson. Summary of anterior-posterior axis formation The AP axis is specified by three sets of genes: Genes that define the anterior organizing center. Bicoid and Hunchback Genes that define the posterior organizing center. Nanos and Caudal Genes that define the terminal boundary regions. Tailless and Huckebein  Figure 1: Practice problem You’ve found a mutant embryo that lacks Bicoid protein, and has developed without anterior head structures (figure 1). You sequence the embryo and find a mutation in a gene called Serendipity. This same homozygous mutation is present in the mother of this embryo. To investigate the role of Serendipity further, you Figure 2: tag Serendipity with GFP to visualize the protein in the developing egg chambers (figure 2). Based on these results: What is missing from mutated Serendipity that causes its change in localization? Come up with a hypothesis that explains Serendipity’s role in Bicoid protein accumulation. Segmentation genes After the maternal effect genes set up the anterior-posterior axis, the zygotic segmentation genes are activated. Segmentation genes are named and categorized based on their mutant phenotypes: Gap genes: mutants lack large regions of the body (several contiguous segments) Pair-rule genes: mutants lack portions of every other segment Segment polarity genes: mutants show defects (deletions, duplications, polarity reversals) in every segment  Practice question Wild-type larva: Mutant 1: Mutant 2: Mutant 3: Which mutant is a gap mutant, which is a pair-rule mutant, and which is a segment polarity mutant?  Practice question Wild-type larva: Mutant 1: Mutant 2: Mutant 3: Pair-rule gene mutant Gap gene mutant Segment polarity gene mutant Gap genes * Gap genes are expressed in broad, overlapping domains that span several segments. Gap gene mutants lack large regions of the body (several contiguous segments). Gap gene expression is induced by the maternal effect mRNAs (Bicoid, Hunchback, Caudal). Expressed by the end of the 12th nuclear division. Pair-rule genes ~ Pair-rule genes are expressed at the boundaries between segments. In pair-rule mutants, the number of segments is reduced. Pair-rule genes are activated by gap genes. Expressed between the 12-13th nuclear divisions. Segment polarity genes Segment polarity genes are expressed in some region of every segment (e.g. the posterior end of every segment). Segment polarity gene mutants show defects (deletions, duplication, polarity reversals) in every segment. Their expression is induced by pair-rule genes. Expression is around the 13th-14th nuclear division, after pair-rule genes. Expression of all segmentation genes is established before gastrulation. Segmentation genes Bargiela et al. 2021. Practicing logical reasoning through Drosophila segmentation gene mutants. Biochemistry and Molecular Biology Education. Segmentation genes *don’t need to know all these names. Just the specific examples that we go over in class. Segments and parasegments pair-rule gene expression: fushi The first indication of segmental periodicity in the tarazu (ftz) is expressed in seven stripes across the embryo Drosophila embryo is pair-rule gene expression. Developmental biologists assumed that these stripes would correspond to the physical segments they saw in later stages of embryogenesis. This isn’t the case. Pair-rule genes are expressed in “parasegmental” domains that straddle the future segmental Physical segments in the borders. Drosophila embryo  Segments and parasegments Head segments: Ma: mandibular Mx: maxillary Ma: labial T1-T3: thoracic segments A1-A8: abdominal segments Segments Ma Mx Lb T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8  Segments and parasegments Each segment has an anterior (A) and a posterior (P) compartment. Segments Ma Mx Lb T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8 Compartments A P A P A PA P A P A PA P A P A P A P A P A PA P A P  Segments and parasegments - Each parasegment consists of the posterior compartment of one segment and the anterior compartment of the segment in the next posterior position. Segments Ma Mx Lb T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8 Compartments A P A P A PA P A P A PA P A P A P A P A P A PA P A P Parasegments 1 2 3 4 5 6 7 8 9 10 11 12 13 14  Segments and parasegments Segments and parasegments are out of phase by one compartment. Segments Ma Mx Lb T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8 Compartments A P A P A PA P A P A PA P A P A P A P A P A PA P A P Parasegments 1 2 3 4 5 6 7 8 9 10 11 12 13 14 fushi tarazu expression  Segments and parasegments = The parasegment is the fundamental - unit of embryonic gene expression. Parasegmental gene expression is not seen in the adult epidermis or the musculature.