Chapter 9 Part 1 | The Genetics of Axis Specification in Drosophila 1. Compare the process of Drosophila fertilization to that of sea urchins and mammals in terms of 1) site of sperm entry, 2) prevention of polyspermy. ○ Site of sperm entry: micropyle ○ Prevention of polyspermy: only one sperm can enter the micropyle *Note: the sperm enters the egg intact (no membrane fusion), egg is activated BEFORE fertilization 2. Describe the type of cleavage and mode of specification of Drosophila embryos Superficial cleavage: single layer of cells encloses the yolky center 3. Explain how nuclei are found within the syncytial blastoderm and how the distribution of nuclei is critical for cell fate determination via exposure to morphogen gradients Syncytium: nuclei divide without cytokinesis Nuclei occupy a defined space (energids) Microtubules ensure the nuclei retain their position as they divide 4. Describe the major events of the mid-blastula transition ○ Nuclear division slows down (gap phases) ○ Cellularization ○ Enhanced transcription of the zygote’s genome ○ Degradation of maternal mRNAs ○ Switch from maternal effect genes to zygotic genes ○ microRNA transcribed at higher rates 5. Explain how the anterior-posterior axis of Drosophila is specified prior to fertilization ○ i.e. where do the factors that specify the A-P axis come from and how do they become localized to different regions of the oocyte? Hierarchy of genes that establish the A-P axis and divide the embryo into segments that have unique identity and polarity Invagination of the mesoderm Invagination of the endoderm anteriorly and posteriorly Convergent extension: germ band formation that gives rise to the thoracic and abdominal segment Migration of pole cells 6. Identify the major classes of genes (i.e., maternal, gap, pair-rule, segment-polarity, and homeotic) that specify the anterior-posterior axis of Drosophila, and describe their the temporal order of expression. ○ Maternal effect genes: deposited into the oocyte prior to fertilization expressed from maternal mRNA’s that form protein gradients i. Transcription factors and transcription regulators that activate GAP genes ○ GAP genes: Transcription factors expressed in broad regions of the embryo i. Mutation: larva lacks multiple contiguous segments ○ Pair-rule genes: Transcription factors i. Divide the embryo into 7 segments and activate the segment polarity genes ○ Segment polarity genes: paracrine factors that divide the embryo into 14 segments ○ Homeotic genes: gives each segment its specific identity (activated by the first 4) 7. Identify the pattern of expression, major functions, and regulatory mechanisms of the maternal effect genes. See above 8. Describe how the maternal-effect genes set up the anterior-posterior axis of Drosophila Occurs prior to fertilization The oocyte is enclosed in an egg chamber, one of the 16 cells derived from the oogonium that divides 4 times producing 16 cells→ 15 become nurse cells and 1 becomes the oocyte located posterior relative to the nurse cells. Cytokinesis b/w nurse cells and oocyte is incomplete, therefore there are connection b/w the cells that allow for the transport of mRNAs along microtubules. Follicular cells surround nurse cells and the oocyte, making up the egg chamber. Transport of mRNA for the protein Gurken to the oocyte Gurken is then translated into protein in the oocyte, localizes b/w the nucleus and cells that will become the posterior follicle cells of the egg chamber (do not form part of the embryo) when Gurken binds to these cells the follicle cells respond by secreting proteins into the oocyte that re-arrange the microtubules so that all the fast-growing ends (+ end) face to the future posterior, while all the slow-growing ends (- end) face towards the future anterior. Important for the localization of mRNAs that will be transported from the nurse cells, since motor proteins that move along the microtubules move towards the + end or towards the end. Ex: Bicoid mRNA associates with the microtubule motor protein dynein moving towards the - end and since all the - end face the future anterior, bicoid will become localized in the future anterior. Other mRNAs are also deposited from the nurse cells into the oocyte, mRNA for Nanos (posterior structures). Oskar first has to be transported to the posterior region, since Oskar associates with Kinesin I which moves towards the + end, trapping Nanos in this location. 9. Explain the experimental evidence that suggests that Bicoid acts as a morphogen and be able to predict the phenotype of fly embryos injected with Bicoid mRNAs in different locations Bicoid and Nanos are translated during ovulation (right before fertilization) and since they are not trapped, they diffuse form their site of synthesis → forming gradients with bicoid being highest at the anterior and bicoid lowest at the posterior. Lose it and move it strategy If an embryo is taken from a bicoid mutant (mother cannot pass bicoid to the oocyte) → the oocyte lacks all anterior structures, instead has two tails that flank an abdomen. Bicoid differentiates b/w acorn and tail since it’s not present two tails form! If bicoid is added to this mutant the normal phenotype is rescued, if added in the center then a head forms in this location, and no acorn is formed since the genes required to form acorn are only expressed at the most anterior/posterior end. When bicoid is injected in the center it is flanked by the thorax, suggesting that bicoid acts as a morphogen. At higher bicoid concentrations the genes required to form the head are activated, at lower concentrations, the genes required to form the thorax are activated. If bicoid is injected into the posterior of a wildtype embryo a second acorn forms followed by a head and a thorax. The signaling pathway needed to form the acorn is the same as the one for the telson. 10. Briefly explain how the terminal-gene group aids in the specification of the unsegmented extremities of the anterior and posterior and, given that the same terminal gene groups are expressed at each extremity of the Drosophila embryo, explain what factor determines the identity (acron or telson) of the segment (this will also be covered in the next lecture). 11. Explain how the maternal effect genes act both transcriptionally and translationally to specify the anterior-posterior axis. The localization of the bicoid mRNA to the future anterior is dependent on its 3’ UTR, if it is altered this would affect the location and as a result the fate of the anterior segments. Also important for the localization of Nanos, the 3 UTR allows it to interact with Oskar. As Nanos diffuses it is inhibited from being translated into protein by inhibitors that bind to its 3’ UTR. Bicoid is actively transported along the microtubules whereas Nanos diffuses to the posterior and becomes trapped by the Oskar (which was transported along the microtubules) Bicoid and Nanos mRNA remain dormant until right before fertilization whn hormonal changed lead to the unmasking of mRNAs. The mRNAs are translated into protein → diffuse forming a gradient so that different nuclei become exposed to different concentrations. Hunchback: important for anterior structures Caudal: posterior structure Note: Found throughout the entire egg, so how are they differentiated? After fertilization, once the bicoid and Nanos are translated into proteins they are not only acting as TF’s but also translation inhibitors. In the anterior bicoid inhibits caudal, in the posterior Nanos blocks hunchback. Bicoid: TF that functions as a morphogen + translation regulator Morphogens: secreted proteins (does not happen if a protein is a TF since they are not secreted but in the Drosophila embryo there is a syncytium and therefore different nuclei can become exposed to different concentrations) Hunchback (maternal effect + GAP gene) activated by hunchback → together they activate? Chapter 9 Part 2 | The Genetics of Axis Specification in Drosophila 1. Describe the phenotypes of gap, pair-rule and segment-polarity mutants and explain how these phenotypes correlate with the expression patterns of these genes The second stage of cell fate commitment is Determination of which cell fate is no longer flexible is mediated by a large group of genes collectively known as the segmentation genes and those include the gap gene, the Pair-Rule genes and the Segment polarity genes. Para-zygotic segmentation of genes: 1.Gap: The gap genes are the first to be express in the Drosophila embryo and the first zygotic gene. Gap genes are known as gap genes because gap mutants lack large portion of the body extending several segments. The reason for this is that Gap genes are express in one or two broad regions of the body and those regions are composed of multiple segments. 2. Pair-Rule Genes: Pair rule gene mutants are missing portions of every other segment, the reason for this is because these genes are expressed in pair segments which are composed of the posterior region or compartment of one segment AND the anterior compartment of the next segment. 3. Segment Polarity: Segment polarity mutants have different types of defects in every segment; this could be an inversion in the polarity, a duplication, or a deletion of the segment. 2. Developmental genes act hierarchically during pattern formation, first defining broad regions, and then smaller ones. Summarize how this general principle is illustrated by early Drosophila embryogenesis. There are 4 major gap genes which include the gene hunchback (Hb) which is also a maternal effect gene as well as the gene Kr, Gt, and Kni. If all four of these genes were deleted the entire segmented portion of the fly would be missing since each of these genes are expressed in broad regions of the fly. The expression of the different gap genes is determined by the maternal effect; so bicoid (Bcd) and caudal (Cad) and the hunchback protein that was transcribed from the maternal mRNA are important for initiating the expression of the different gap genes. The gap genes also regulate each other. Bicoid is located in the anterior region of the embryo acts as an activator for the genes hunchback (Hb) , Kni, and Gt. Caudal also acts as an activator for giants (Gt); so Gt is expressed in 2 different regions along the anterior-posterior axis of the embryo; its expression in the anterior of the embryo is activated by bicoid; while the expression of the posterior is activated by Caudal. The reason for Gt is expressed in both sections is because of enhancers; enhancers determines when and where a particular gene is expresses so Gt can have different enhancers for different regions. The expression of the gap genes is initiated by the maternal effect genes, but it is maintained by the interaction between the different gap genes because these are transcription factors and because at this point the embryo is still a syncytium. They’re still nuclei that are sharing the same cytoplasm. The regulation among the different gap genes, the hunchback genes is expressed in a broad region at the anterior of the embryo. Hb and Kni inhibits each other expressions; so Kni expression only increases where the level of maternal and zygotic hunchback begin to drop along the anterior-posterior axis of the embryo. At high concentrations Hb acts also as an inhibitor for Kr gene; but Kruppel (Kr) is also activated by lower concentrations of Hb; this is why its expression increases about halfway through the embryo where the concentrations of hunchback begin to decline. Kr and Gt interact also, they act as repressors of each other. The interaction between the different gap genes results in a pattern of mRNA expression where there is overlap between the mRNA’s of these different genes; As the mRNAs are translated the proteins diffuse from their side of synthesis and this contribute to regions of overlap among the different gap proteins. This gene also contains redundant enhancers, so they contain multiple enhancers that drive expression to the same region. 3. Identify the major segments of the Drosophila larva and adult and explain how these relate to parasegments? 4. Describe how the expression patterns of the gap genes are initiated and maintained. 5. What do the gap genes do? (Hint: they activate other genes). 6. Identify the general cis and trans regulatory elements that regulate the expression of pair-rule genes and explain how different enhancers drive expression of the same pair-rule gene in different stripes along the anterior-posterior axis. 7. Explain how the proteins coded by the segment-polarity genes differ from proteins coded by the maternal, gap, and pair-rule genes and explain why this is important, keeping in mind that the timing of segment-polarity gene expression is after cellularization. 8. Explain how the pattern of expression of the segment polarity genes are initiated and maintained? What allows these genes to be expressed throughout the lifetime of the organism? 9. What is the role of the segment polarity genes and how is this role achieved? 10. Describe how the molecular organization of the homeotic selector genes corresponds to their patterns of expression. 11. Discuss the function of the homeotic selector genes and the types of phenotypes that arise from alterations in these genes 12. Describe the process used to specify the dorsal-ventral axis of Drosophila, explaining how Dorsal functions as a morphogen despite being expressed throughout the entire embryo. 13. Describe the phenotypes that would result from mutations that inactivate Dorsal or its ability to enter cell nuclei 14. Describe the phenotypes of mutations that allow Dorsal to enter all cell nuclei. Chapter 11 | Amphibian Development 1. Identify the pattern of cleavage of amphibian embryos and explain the reason why cleavage at the vegetal pole happens more slowly compared to cleavage at the animal pole (we have also covered this in previous lectures) 2. Describe the events that happen as a result of sperm entry (i.e. microtubule rearrangement, cortical rotation) and explain the effect of these events on the specification of the dorsal-ventral axis. 3. Describe the developmental significance of the gray crescent (what is the gray crescent, where is it, and what is so special about it?) 4. Explain how and why the blastocoel plays a role in the specification of the germ layers. 5. Identify the major events of the mid-blastula transition. 6. Identify the site where gastrulation begins in amphibian embryos and explain how this site relates to the site of sperm entry. 7. Describe the general cell movements that occur during amphibian gastrulation and explain the mechanism of convergent extension and the role of fibronectins in gastrulation (review what fibronectin is (Chapter 4) and explain why gastrulation would be affected if cells were prevented from binding to fibronectins? 8. Examine how the polarity of the amphibian oocyte (in terms of maternal mRNA distribution) plays a role in establishing the germ layers. 9. Identify the tissues that make up the amphibian organizer and explain why these tissues have become known as ‘the organizer’. 10. Explain how the Wnt signaling pathway, particularly β-catenin, contributes to the specification of multiple axes in the amphibian embryo 11. Describe how the interactions between the mesoderm and ectoderm, which happen during gastrulation, help specify the tissues of the nervous system along the anterior-posterior axis. 12. For each of the experiments carried out by Mangold and/or Spemann, describe the experiment and explain what the results showed. For example, for the experiment in which Spemann used a hair to separate the cytoplasm of the newt blastula after fertilization, why did he obtain different results depending on how he separated the zygote (along the plane of the first cleavage vs. perpendicular to the plane of the first cleavage)? 13. Describe the properties/functions of the amphibian organizer.
