1!Gastrulation III - establishment of body axes • Anterior-Posterior Axis Formation in Drosophila.!– Bicoid, the molecular interpretation of a gradient!• Anterior-Posterior Axis Formation in Mouse.!– Formation of the primitive streak!– Anterior Visceral Endoderm!Gastrulation III - establishment of body axes • Anterior-Posterior Axis Formation in Drosophila.!– Bicoid, the molecular interpretation of a gradient!• Anterior-Posterior Axis Formation in Mouse.!– Formation of the primitive streak!– Anterior Visceral Endoderm! Drosophila malenogaster Fly Anatomy!Egg! First Instar Larva!Anterior! Posterior!The Axis is Established Through a Graded Reduction in Pattern! Drosophila malenogaster The Axis is Established Under "Maternal Control!Mutations Affecting the "Anterior-Posterior Axis!Anterior-Posterior Axis Formation is Independent of Dorsal-Ventral Axis Formation.!Bicoid (Bcd) Controls Drosophila" A-P Patterning !Bcd!Hb!DAPI!2!Evidence the Bcd is the Key Regulatory Gene!1. Strong Bcd alleles lead to a complete loss of head structures. !2. Bcd mutants can be completely rescued by injection of wild-type anterior cytoplasm.!3. Size of head directly related to Bcd gene dosage. !Mutation in Bcd lead to a complete loss of head structures!Bcd is Required for the Formation of Anterior Structures!Phenocopies Removal of Anterior Cytoplasm!Evidence the Bcd is the Key Regulatory Gene!1. Strong Bcd alleles lead to a complete loss of head structures. !2. Bcd mutants can be completely rescued by injection of wild-type anterior cytoplasm.!3. Size of head directly related to Bcd gene dosage. !Transplantation of Wildtype Anterior Cytoplasm Can Rescue Bcd Mutants!Transfer of Bcd RNA is Sufficient to Induce Anterior Structures"Bcd May Act as a Morphogen!Evidence the Bcd is the Key Regulatory Gene!1. Strong Bcd alleles lead to a complete loss of head structures. !2. Bcd mutants can be completely rescued by injection of wild-type anterior cytoplasm.!3. Size of head directly related to Bcd gene dosage. !Hypothesis: Bcd establishes the anterior-posterior axis through the establishment of a morphogen gradient."Morphogen!An inducing factor that can evoke more than one cell state from the responding tissue.3!Gradient"The asymmetric distribution of a protein or protein activity in a tissue.!Hypothesis: Bcd establishes the anterior-posterior axis through the establishment of a morphogen gradient."Prediction: Bcd must be a cytoplasmic determinant localized to the anterior pole. !Establish A-P axis through localization of Bcd RNA and protein in anterior pole!Establish A-P axis through localization of Bcd RNA and protein in anterior pole!Posterior pole must be cleared of hunchback by nanos.!Posterior: Posterior pole must "be cleared of hunchback!Main Function of nanos is to Block Hunchback!How does the embryo interpret the Bcd gradient?!The Conversion of Maternal Information to Zygotic readout.!The Maternal Systems That Establish "A-P Position in the Drosophila Egg Activate Exclusively "Zygotic Transcription Factors!4! Drosophila malenogaster Quantitative Relationship Between the Number of Bcd and the Pattern of the Embryo!Bcd can Activate and Repress Target Genes at Defined Thresholds!The Axis is Established Through a Graded Reduction in Pattern!Gastrulation III - establishment of body axes • Anterior-Posterior Axis Formation in Drosophila.!– Gradients and Morphogenesis!– Bicoid!• Anterior-Posterior Axis Formation in Mouse.!– Formation of the primitive streak!– Anterior Visceral Endoderm!Specification ≠Fate!Hypothesis: Must exist region a third signal that modifies mesodermal cell types.!The 3-Signal Model (Smith and Slack)!Dorsal!Animal!Vegetal!Ventral!Ectoderm!Mesoderm!Endoderm!3-Signal Model Circa 2008!Wnt! Mus Musculus domesticus5!Drosophila vs. Xenopus!Xenopus vs. Mouse!Mouse Node!Node verses Organizer!• Transplantation of either to distal regions of the embryo can lead to axis (a-p and d-v) formation.!• Both ultimately will give rise to notochord and gut endoderm!• Both the organizer and the node express many of the same genes.!• The organizer but NOT the node will induce anterior structures; i.e. head!• The organizer is intimately linked with the blastopore lip spatially and temporally the organizer is not linked with the primitive streak spatially and temporally !Node verses Organizer!• Transplantation of either to distal regions of the embryo can lead to axis (a-p and d-v) formation.!• Both ultimately will give rise to notochord and gut endoderm!• Both the organizer and the node express many of the same genes.!• The organizer but NOT the node will induce anterior structures; i.e. head!• The organizer is intimately linked with the blastopore lip spatially and temporally the organizer is not linked with the primitive streak spatially and temporally !Conclusion: Must be other source of organizing signals responsible for anterior structures in the mouse.!The AVE is Required for Proper Anterior Patterning!AVE Patterns the Anterior "Portion of the Embryo!Embryonic Ecotoderm!Visceral Endoderm!AVE!Formation of the AVE!6!Does gene expression in the AVE correlate with anterior organizing function?!Evidence!1. Transplant mouse node get a duplicated axis but secondary axis lacks a head. !2. Genes found to be expressed exclusively within the AVE prior to primitive streak formation.!3. Remove AVE headless embryos results or add AVE to epiblast tissue not normally giving rise to anterior structures induces expression of anterior genes. !4. Mutation in genes expressed in AVE (e.g. Hesx1, Lim1, Otx2) associated with anterior truncations.!5. Cripto -/- mice lack an organizer but retain some anterior patterning.!6. Removal of nodal only from the VE results in anterior truncations.!Removal of nodal only from the VE results in anterior truncations!Basis of experiments:!!1. Decedents of tetraploid blastocysts only give rise to extraembryonic tissue.!!2. ES cells injected into blastocysts give little to no contribution to the visceral endoderm and trophectoderm.!Restriction of Gene expression Patterns in AVE Precedes Streak Formation!AVE itself can be subdivided molecularly into head verses heart inducing tissue!Chimeric Studies to Test the Requirement for nodal in the AVE!AVE Patterns the Anterior "Portion of the Embryo!Embryonic Ectoderm!Visceral Endoderm!AVE!ES Cell