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1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Adult Cell Cycle Cell Cycle of Early Frog Blastomere Mitosis Active Cdk / G2 cyclin M C G1 Cdk / G1 cyclin Cdk / cyclin Active Mitosis C M G2 M Active Cyclin Synthesis Cdk / Active S cyclin S DNA Synthesis a. S G2 interphase M mitosis C cytokinesis Cyclin Degradation S Inactive S Cdk DNA Synthesis b. 2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nematode Lineage Map Egg Egg and sperm line Nervous system Pharynx Intestine Cuticle-making cells Vulva Gonad a. Adult Nematode Gonad Gonad Intestine Nervous system Sperm Egg Cuticle Pharynx Vulva b. 3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Normal Not Determined (early development) Determined (later development) No donor Donor Tail cells are transplanted to head Tail cells are transplanted to head Recipient Before Overt Differentiation Tail Head Recipient After Overt Differentiation Tail cells develop into head cells in head Tail cells develop into tail cells in head 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. MEIOSIS Sperm (haploid) n 50 µm Egg (haploid) n Adult tunicate (diploid) 2n n 50 µm 2n Pigment granules Embryo (diploid) 2n 50 µm Larva (diploid) 2n 50 µm a. b. b: © J. Richard Whittaker, used by permission 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Sagittal section 1 Longitudinal section 1 2 Anterior Anterior Posterior Dorsal nerve cord (NC) Notochord (Not) Ventral endoderm (En) Longitudinal section 2 Mesenchymal cells (Mes) Posterior Tail muscle cells (Mus) a. FGF signaling Anterior Posterior 32-Cell Stage b. 64-Cell Stage c. 6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. First Step Second Step Cell Types Yes Yes No Mesenchyme FGF Signal received No FGF Signal received Yes No Notochord Macho-1 inherited? Muscle Nerve cord a. No FGF FGF FGF Receptor FGF Receptor FGF Receptor FGF Receptor Cell membrane Cell membrane Cell membrane Cell membrane Ras/MAPK Pathway Ras/MAPK Pathway T-Ets No FGF FGF Macho-1 T-Ets Macho-1 P Ras/MAPK Pathway Ras/MAPK Pathway T-Ets T-Ets No Macho-1 No Macho-1 P Suppression of muscle genes and activation of mesenchyme genes Mesenchyme Precursor Cells Transcription of muscle genes Muscle Precursor Cells Transcription of notochord genes Notochord Precursor Cells Suppression of notochord genes and activation of nerve cord genes Nerve cord Precursor Cells b. 7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Once sperm cell and egg cell have joined, cell cleavage produces a blastocyst. The inner cell mass of the blastocyst develops into the human embryo. Embryonic stem cell culture Inner cell mass Egg Sperm Blastocyst Embryo Embryonic stem cells (ES cells) are isolated from the inner cell mass a. b. 500 µm b: © University of Wisconsin-Madison 8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Preparation Cell Fusion Mammary cell is extracted and grown in Mammary cell is nutrient-deficient solution that arrests the cell cycle. inserted inside Nucleus containing covering of egg cell. Cell Division Electric shock fuses cell membranes and triggers cell division. Development Embryo begins to develop in vitro. source DNA Egg cell is extracted. Implantation Birth of Clone Growth to Adulthood Embryo is After a five-month pregnancy, a implanted into lamb genetically identical to the surrogate sheep from which the mammary mother . cell was extracted is born. Nucleus is removed fro egg cell with a micropipette. Embryo © APTV/AP Photo 9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Oocyte Nuclear Transfer Somatic cells Somatic cells Fusion Blastocyst ES cells Defined factors Culture Pluripotent stem cells Somatic cells Germ cells Some adult stem cells 10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The nucleus from a skin cell of a diabetic patient is removed. The skin cell nucleus is inserted into the enucleated human egg cell. Cell cleavage occurs as the embryo begins to develop in vitro. The embryo reaches the blastocyst stage. Therapeutic cloning Embryonic stem cells (ES cells) are extracted and grown in culture. The stem cells are developed into healthy pancreatic islet cells needed by the patient. The healthy tissue is injected or transplanted into the diabetic patient. Inner cell mass Diabetic patient Healthy pancreatic islet cells Early embryo ES cells Blastocyst Diabetic patient 11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Movement of maternal mRNA Follicle cells Nurse cells Posterior Oocyte Anterior Nucleus Fertilized egg a. Syncytial blastoderm Nuclei line up along surface, and Cellular blastoderm membranes grow between them to form a cellular blastoderm. Segmented embryo prior to hatching b. Hatching larva Three larval stages c. Metamorphosis d. Thorax Head Abdomen e. 12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Establishing the Polarity of the Embryo Fertilization of the egg triggers the production of Bicoid protein from maternal RN A in the egg. The Bicoid protein diffuses through the egg, forming a gradient. This gradient determines the polarity of the embryo, with the head and thorax developing in the zone of high concentration (green fluorescent dye in antibodies that bind bicoid protein allows visualization of the gradient). Setting the Stage for Segmentation About 21/2 hours after fertilization, Bicoid protein turns on a series of brief signals from so-called gap genes. The gap proteins act to divide the embryo into large blocks. In this photo, fluorescent dyes in antibodies that bind to the gap proteins Krüppel (orange) and Hunchback (green) make the blocks visible; the region of overlap is yellow. 500 µm 500 µm Laying Down the Fundamental Regions About 0.5 hr later , the gap genes switch on the “pair-rule” genes, which are each expressed in seven stripes. This is shown for the pair-rule gene hairy . Some pair-rule genes are only required for even-numbered segments while others are only required for odd numbered segments. 500 µm Forming the Segments The final stage of segmentation occurs when a “segmentpolarity” gene called engrailed divides each of the seven regions into halves, producing 14 narrow compartments. Each compartment corresponds to one segment of the future body . There are three head segments (H, bottom right), three thoracic segments (T, upper right), and eight abdominal segments (A, from top right to bottom left). A a: © Steve Paddock and Sean Carroll; b-d: © Jim Langeland, Steve Paddock and Sean Carroll T H 500 µm 13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Movement of bicoid mRNA moves maternal mRNA toward anterior end Nurse cells Nucleus Follicle cells Anterior Anterior Posterior Microtubules nanos mRNA moves toward posterior end a. Posterior bicoid mRNA nanos mRNA b. 14 Concentration Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. nanos mRNA hunchback mRNA bicoid mRNA caudal mRNA Posterior Anterior a. Oocyte mRNAs Anterior bicoid mRNA Bicoid protein caudal mRNA Caudal protein Posterior nanos mRNA Nanos protein hunchback mRNA Hunchback protein Concentration b. After fertilization Nanos protein Hunchback protein Bicoid protein Caudal protein Anterior c. Early cleavage embryo proteins Posterior 15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a. 400 µm b. 400 µm Dorsal Wild-type embryo Ventral c. dorsal mutant 100 µm a: © Dr. Daniel St. Johnston/Wellcome Images; b: © Schupbach, T. and van Buskirk, C.; c: From Roth et al., 1989, courtesy of Siegfried Roth 16 17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Drosophila HOM Chromosomes Drosophila HOM genes Antennapedia complex lab pb Dfd Scr Antp Head Thorax Mouse Hox Chromosomes Hox 1 Bithorax complex Ubx abd-A abd-B Hox 2 Hox 3 Hox 4 Abdomen Fruit fly embryo Mouse embryo Mouse Fruit fly a. b. 18 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Organism Caenorhabditis elegans Inhibitor: CED-9 Activator: CED-4 Apoptotic Protease: CED-3 Mammalian Cell Inhibitor Apaf1 Caspase-8 or -9 Apoptosis Inhibition Activation a. Bcl-2 Apoptosis b. 19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. SCIENTIFIC THINKING Hypothesis: Fibronectin is required for cell migration during gastrulation. Prediction: Blocking fibronectin with antifibronectin antibodies before gastrulation should prevent cell movement. Test: Staged salamander embryos were injected either with antifibronectin antibody, or with preimmune serum as a control, prior to gastrulation. Cell movements were then monitored photographically. Treated with Preimmune Treated with Antifibronectin Blastopore Cells have moved 285.7 µm into the interior a. Blastopore Cells pile up on the surface 285.7 µm b. Result: The experimental embryos injected with antifibronectin antibody show extremely aberrant gastrulation where cells pile up and do not enter the interior of the embryo. Control embryos gastrulate normally. Conclusion: Fibronectin is required for cells to migrate into the interior of the embryo during gastrulation. Further Experiments: How can this same system be used to analyze the role of fibronectin in other early morphogenetic events? a-b: From Boucaut et al., 1984, courtesy of J-C Boucaut. 20 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Epidermal cells Shoot apical meristem Ground tissue cells Vascular tissue cells Embryo Embryo Suspensor Cotyledons a. Early cell division b. Tissue formation Shoot apical meristem Seed wall Cotyledons Root apical meristem Root apical meristem c. Seed formation d. Germination e. Meristematic development and morphogenesis 21