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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. C M G 2 interphase mitosis cytokinesis G2G2 S G1G1 M C Mitosis Adult Cell Cycle S Mitosis Active Cell Cycle of Early Frog Blastomere Active C M S S M Cdk Inactive a.b. Cdk / G1 cyclin Cdk / G2 cyclin Cdk / S cyclin G1G1 DNA Synthesis Cyclin Degradation Cyclin Synthesis Cdk / cyclin DNA Synthesis 2

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nematode Lineage Map a. b. Egg Pharynx Cuticle-making cells Egg Sperm Adult Nematode Vulva Gonad Nervous system Pharynx Intestine Cuticle Intestine Egg and sperm line Nervous system Vulva 3

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Donor No donor Normal Head Recipient Before Overt Differentiation Recipient After Overt Differentiation Tail cells develop into head cells in head Tail cells develop into tail cells in head Tail cells are transplanted to head Tail cells are transplanted to head Not Determined (early development) Determined (later development) Tail 4

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. MEIOSIS a. n 2n b. FERTILIZATION 50 µm b: © J. Richard Whittaker, used by permission Adult tunicate (diploid) 2n Larva (diploid) 2n Embryo (diploid) 2n Pigment granules Sperm (haploid) n Egg (haploid) n 5

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. b.c. 2 1 Notochord (Not) Anterior Posterior AnteriorPosterior a. 21 FGF signaling Sagittal sectionLongitudinal section Dorsal nerve cord (NC) Mesenchymal cells (Mes) 32-Cell Stage64-Cell Stage Anterior Posterior Ventral endoderm (En) Tail muscle cells (Mus) 6

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. b.b. FGF Receptor No FGF FGF Receptor No FGF FGF Receptor Cell membrane Ras/MAPK Pathway T-Ets No Macho-1 T-Ets No Macho-1 T-EtsMacho-1 Suppression of muscle genes and activation of mesenchyme genes Transcription of muscle genes Transcription of notochord genes Nerve cord Precursor Cells Notochord Precursor Cells Muscle Precursor Cells Mesenchyme Precursor Cells a.a. P P Suppression of notochord genes and activation of nerve cord genes Cell membrane Mesenchyme Muscle Notochord FGF Signal received Cell TypesSecond StepFirst Step Macho-1 inherited? Yes No Nerve cord Yes No Yes No FGF 7

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a.b. Egg SpermBlastocystEmbryo Embryonic stem cells (ES cells) are isolated from the inner cell mass Embryonic stem cell culture 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. Inner cell mass b: © University of Wisconsin-Madison 500 µm 8

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © APTV/AP Photo Embryo Egg cell is extracted. Development ImplantationBirth of Clone PreparationCell Fusion Cell Division Mammary cell is extracted and grown in nutrient-deficient solution that arrests the cell cycle. Nucleus containing source DNA Nucleus is removed fro egg cell with a micropipette. Mammary cell is inserted inside covering of egg cell. Electric shock fuses cell membranes and triggers cell division. Embryo begins to develop in vitro. Embryo is implanted into surrogate mother. After a five-month pregnancy, a lamb genetically identical to the sheep from which the mammary cell was extracted is born. Growth to Adulthood 9

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Somatic cells Oocyte Somatic cells Blastocyst Fusion Culture Somatic cells ES cells Nuclear Transfer Pluripotent stem cells Defined factors Germ cells Some adult stem cells 10

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Early embryo Blastocyst 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. Inner cell mass ES cells 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. Healthy pancreatic islet cells Therapeutic cloning Diabetic patient Diabetic patient The nucleus from a skin cell of a diabetic patient is removed. 11

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hatching larva c. b. AnteriorPosterior Oocyte Fertilized egg Nucleus a. d. e. Three larval stages Syncytial blastoderm Cellular blastoderm Segmented embryo prior to hatching Metamorphosis Abdomen Thorax Head Movement of maternal mRNA Follicle cells Nurse cells Nuclei line up along surface, and membranes grow between them to form a cellular blastoderm. 12

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Forming the SegmentsLaying Down the Fundamental Regions Setting the Stage for SegmentationEstablishing the Polarity of the Embryo H T A 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. The final stage of segmentation occurs when a “segment- polarity” 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). 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 a: © Steve Paddock and Sean Carroll; b-d: © Jim Langeland, Steve Paddock and Sean Carroll 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). 13

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a. b. Nucleus Microtubules PosteriorAnterior PosteriorAnterior Movement of maternal mRNA bicoid mRNA moves toward anterior end Follicle cells nanos mRNA moves toward posterior end Nurse cells bicoid mRNA nanos mRNA 14

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Concentration Anterior a. Oocyte mRNAs c. Early cleavage embryo proteins b. After fertilization Posterior Nanos protein Hunchback protein Bicoid protein Caudal protein Concentration AnteriorPosterior Nanos protein Hunchback protein Bicoid protein Caudal protein nanos mRNA hunchback mRNA bicoid mRNA caudal mRNA hunchback mRNA bicoid mRNA caudal mRNA nanos mRNA 15

a. b. c. Dorsal dorsal mutant 400 µm 100 µm Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a: © Dr. Daniel St. Johnston/Wellcome Images; b: © Schupbach, T. and van Buskirk, C.; c: From Roth et al., 1989, courtesy of Siegfried Roth Wild-type embryoVentral 16

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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Drosophila HOM genes Thorax Antennapedia complex HeadAbdomen Bithorax complex Fruit fly Mouse Hox 1 Hox 2 Hox 3 Hox 4 a.b. Drosophila HOM Chromosomes Mouse Hox Chromosomes labpbDfdScrAntpUbxabd-B Mouse embryo Fruit fly embryo abd-A 18

a.b. InhibitorCED-9Bcl-2 CED-4Apaf1 Caspase-8 or -9CED-3 Inhibitor: Activator: Caenorhabditis elegansMammalian CellOrganism Inhibition Activation Apoptotic Protease: Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Apoptosis 19

a.b. Blastopore 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. 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? Cells have moved into the interior Cells pile up on the surface Treated with Antifibronectin µm Treated with Preimmune SCIENTIFIC THINKING Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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. Embryo Suspensor Seed wall Shoot apical meristem Cotyledons Epidermal cells Ground tissue cells Vascular tissue cells a. Early cell divisionb. Tissue formation Shoot apical meristem Root apical meristem c. Seed formationd. Germinatione. Meristematic development and morphogenesis Root apical meristem 21