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Lecture 5 • Anterior Posterior axis formation: Cell Biology • Bicoid is a morphogen Lab 5 schedule • On Monday and Tuesday come in at the regular time. • On Tuesday and Wednesday come to B&G 3015 to mount your embryos. Elyse will discuss the time of day to come. Exams • Midterm Tuesday Feb. 28, 2012 UCC 63, 7-10PM • Tentative Final exam date Thursday April 19, 2012 at 9:00AM Structure of the egg Dorsal appendage A D P Chorion V Micropyle Vitelline Cellular Blastoderm Hartenstein 1993 Blastoderm fate map Hartenstein 1993 Simplified Fate Map Nusslein-Volhard et al., Science 238, 1675-1681 Classical Embryology Remove 510% of the anterior cytoplasm Remove posterior pole plasm Nusslein-Volhard et al., Science 238, 1675-1681 Transplant anterior cytoplasm to posterior Nusslein-Volhard et al., Science 238, 1675-1681 Transplant posterior cytoplasm to anterior Nusslein-Volhard et al., Science 238, 1675-1681 Transplant posterior cytoplasm to anterior Nusslein-Volhard et al., Science 238, 1675-1681 Transplant posterior cytoplasm to an anterior that lacks the anterior signal Nusslein-Volhard et al., Science 238, 1675-1681 Properties of the polar center signals • Both exert long range effects • The anterior and posterior activities inhibit each other • Both activities have inductive properties Properties of the polar center signals • Both exert long range effects Classical Embryology Remove 510% of the anterior cytoplasm Remove posterior pole plasm Nusslein-Volhard et al., Science 238, 1675-1681 Properties of the polar center signals • The anterior and posterior activities inhibit each other Transplant posterior cytoplasm to anterior Nusslein-Volhard et al., Science 238, 1675-1681 Transplant anterior cytoplasm to posterior Nusslein-Volhard et al., Science 238, 1675-1681 Properties of the polar center signals • Both activities have inductive properties Transplant anterior cytoplasm to posterior Nusslein-Volhard et al., Science 238, 1675-1681 Transplant posterior cytoplasm to an anterior that lacks the anterior signal Nusslein-Volhard et al., Science 238, 1675-1681 What you may know already Bicoid: Anterior signal Nanos: Posterior signal How do we know this? Maternal effect screens Normal egg bcd/bcd mother Larva missing a head Nusslein-Volhard et al., Science 238, 1675-1681 Three classes of maternal effect phenotypes Nusslein-Volhard et al., Science 238, 1675-1681 The real cuticles wt bcd oskar torso Nusslein-Volhard et al., Science 238, 1675-1681 Mutant combinations St Johnston and Nusslein-Volhard Cell 1992 Transplantation of cytoplasm into bcd mutants Nusslein-Volhard et al., Science 238, 1675-1681 Controlling anterior posterior polarity Nusslein-Volhard et al., Science 238, 1675-1681 The difference between the posterior and anterior signals Nusslein-Volhard et al., Science 238, 1675-1681 Distinct Properties of the signals • Anterior signal-instructive. Induces polarity at site of injection. • Posterior signal-permissive. Polarity independent of injection site. Evidence for Bicoid • Rescue activity spatially distributed • Amount of rescue dependent on amount injected • Linear relationship between gene dose and rescue activity • mRNA asymmetrically localized and protein expressed in a gradient Bicoid rescue assay Nusslein-Volhard et al., Science 238, 1675-1681 Rescue activity is spatially distributed 100% % rescue activity 50% 100% 40% % egg length 0% Amount of rescue is dependent on amount injected 100% % rescue activity 50% 0 1/3 2/3 Transplanted volume 1 Linear relationship between gene dose and rescue activity 100% % rescue activity 50% 0 1 2 3 Dose of bicoid gene in mothers mRNA assymetrically localized and protein expressed in a gradient mRNA Protein Driever and Nusslein-Volhard Cell 54, 83-93 Bicoid • Bicoid has the properties of the anterior signal • Does Bicoid need to interact with any other anteriorly localized signals to work? Bicoid is necessary and sufficient Bicoid mRNA made in vitro mRNA injected at posterior pole Bicoid protein expression Larva with two heads Driever et al., 1990 Development 109, 811-820 Bicoid • Bicoid is the anterior signal • What does Bicoid encode? • How does Bicoid work? Bicoid encodes a homeodomain containing protein. Hunchback lacks anterior segments Nusslein-Volhard and Wieschaus 1980 Nature 287, 795 Hunchback is expressed at the anterior pole Bicoid mRNA Bicoid protein Hunchback mRNA expression Driever 1993 Hunchback gene Driever and Nusslein-Volhard Nature 337, 138-143 Bicoid is a transcriptional activator Driever and Nusslein-Volhard Nature 337, 138-143 mRNA assymetrically localized and protein expressed in a gradient mRNA Protein Driever and Nusslein-Volhard Cell 54, 83-93 Morphogen • Long range action • Affect polarity • Concentration dependent • Different fates at different concentrations Morphogen • Long range action Hunchback is expressed at the anterior pole Bicoid mRNA Bicoid protein Hunchback mRNA expression Driever 1993 Morphogen • Affect polarity Bicoid is necessary and sufficient Bicoid mRNA made in vitro mRNA injected at posterior pole Bicoid protein expression Larva with two heads Driever et al., 1990 Development 109, 811-820 Morphogen • Concentration dependent Concentration dependence of Bicoid function Concentration dependence of Bicoid function hb-lacZ expression # of bicoid genes in the mother 0 1 2 4 6 Struhl et al., 1989 Cell 57, 1259-1273 Morphogen • Different fates at different concentrations Distinct Bicoid binding sites are activated at different concentrations of Bicoid Morphogen • Long range action • Affect polarity • Concentration dependent • Different fates at different concentrations How is bicoid mRNA localized to the anterior pole? Other anterior class mutants Driever and Nusslein-Volhard Cell 54, 94-104 Bicoid gradient affected in the mutants Driever and Nusslein-Volhard Cell 54, 94-104 The mutants affect different stages of bicoid mRNA deposition in the developing oocyte St. Johnston et al., 1989 Development sup 13-19 Localization of bicoid mRNA: recent paper Irion and St Johnston 2007 Nature 445, 554-558