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Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? Figure 47.3 The acrosomal and cortical reactions during sea urchin fertilization 1 Contact. The sperm cell contacts the egg’s jelly coat, triggering exocytosis from the sperm’s acrosome. 2 Acrosomal reaction. Hydrolytic enzymes released from the acrosome make a hole in the jelly coat, while growing actin filaments form the acrosomal process. This structure protrudes from the sperm head and penetrates the jelly coat, binding to receptors in the egg cell membrane that extend through the vitelline layer. 3 Contact and fusion of sperm and egg membranes. A hole is made in the vitelline layer, allowing contact and fusion of the gamete plasma membranes. The membrane becomes depolarized, resulting in the fast block to polyspermy. 4 Entry of sperm nucleus. Sperm plasma membrane 5 Cortical reaction. Fusion of the gamete membranes triggers an increase of Ca2+ in the egg’s cytosol, causing cortical granules in the egg to fuse with the plasma membrane and discharge their contents. This leads to swelling of the perivitelline space, hardening of the vitelline layer, and clipping of sperm-binding receptors. The resulting fertilization envelope is the slow block to polyspermy. Sperm nucleus Acrosomal process Basal body (centriole) Fertilization envelope Sperm head Actin Acrosome Jelly coat Sperm-binding receptors Fused plasma Cortical membranes granule Perivitelline Hydrolytic enzymes space Cortical granule membrane Vitelline layer Egg plasma membrane EGG CYTOPLASM Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 1. Contact – acrosome releases hydrolytic enzymes 2. Acrosomal rxn – enzymes digest jelly coat while actin extends - acrosomal process attaches to sperm binding receptors 3. Membrane fusion (sperm & egg) – causes depolarization as Ca+2 released - aka fast block to polyspermy - aka activation of the egg begins 4. Sperm nucleus enters egg 5. Cortical rxn – cortical granules from egg fuse with plasma membrane - Forms fertilization envelope aka slow block to polyspermy 2. What happens with activation of the egg? - Ca+2 released from ER - ↑ Cellular respiration & ↑ protein synthesis (translation) Figure 47.4 What is the effect of sperm binding on Ca2+ distribution in the egg? EXPERIMENT A fluorescent dye that glows when it binds free Ca2+ was injected into unfertilized sea urchin eggs. After sea urchin sperm were added, researchers observed the eggs in a fluorescence microscope. 500 m RESULTS 1 sec before fertilization 10 sec after fertilization Point of Sperm entry 20 sec 30 sec Spreading wave of calcium ions CONCLUSION The release of Ca2+ from the endoplasmic reticulum into the cytosol at the site of sperm entry triggers the release of more and more Ca2+ in a wave that spreads to the other side of the cell. The entire process takes about 30 seconds. Figure 47.5 Timeline for the fertilization of sea urchin eggs 1 Binding of sperm to egg 2 Acrosomal reaction: plasma membrane depolarization (fast block to polyspermy) 3 4 6 8 10 Increased intracellular calcium level 20 Cortical reaction begins (slow block to polyspermy) 30 40 50 1 Formation of fertilization envelope complete 2 Increased intracellular pH 3 4 5 Increased protein synthesis 10 20 Fusion of egg and sperm nuclei complete 30 40 Onset of DNA synthesis 60 90 First cell division Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? Figure 47.6 Early events of fertilization in mammals 1 The sperm migrates through the coat of follicle cells and binds to receptor molecules in the zona pellucida of the egg. (Receptor molecules are not shown here.) 2 This binding induces the acrosomal reaction, in which the sperm releases hydrolytic enzymes into the zona pellucida. 3 Breakdown of the zona pellucida by these enzymes allows the sperm to reach the plasma membrane of the egg. Membrane proteins of the sperm bind to receptors on the egg membrane, and the two membranes fuse. 4 The nucleus and other components of the sperm cell enter the egg. Follicle cell 5 Enzymes released during the cortical reaction harden the zona pellucida, which now functions as a block to polyspermy. Zone pellucida Egg plasma membrane Sperm basal body Cortical Sperm granules nucleus Acrosomal vesicle EGG CYTOPLASM Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? 1. Contact – sperm migrates through follicle cells & binds to zona pellucida 2. Acrosomal rxn – acrosome releases hydrolytic enzymes digesting ZP 3. Sperm bind to sperm receptors on 2° oocyte & membranes fuse 4. Sperm nucleus enters egg 5. Cortical reaction hardens ZP as a block to polyspermy 4. What happens with during cleavage? 1. Cell division w/o cytokinesis 2. Creates blastomeres 3. Axes formed at first cleavage in amphibians Figure 47.8 The body axes and their establishment in an amphibian Anterior (a) Body axes. The three axes of the fully developed embryo, the tadpole, are shown above. Right Ventral Dorsal Left 1 The polarity of the egg determines the anterior-posterior axis before fertilization. Posterior Animal hemisphere Animal pole Point of sperm entry Vegetal hemisphere 2 At fertilization, the pigmented cortex slides over the underlying cytoplasm toward the point of sperm entry. This rotation (red arrow) exposes a region of lighter-colored cytoplasm, the gray crescent, which is a marker of the dorsal side. 3 The first cleavage division bisects the gray crescent. Once the anteriorposterior and dorsal-ventral axes are defined, so is the left-right axis. Point of sperm entry Gray crescent Vegetal pole Future dorsal side of tadpole First cleavage (b) Establishing the axes. The polarity of the egg and cortical rotation are critical in setting up the body axes. Figure 47.9 Cleavage in a frog embryo Zygote 0.25 mm 2-cell stage forming Eight-cell stage (viewed from the animal pole). The large amount of yolk displaces the third cleavage toward the animal pole, forming two tiers of cells. The four cells near the animal pole (closer, in this view) are smaller than the other four cells (SEM). 4-cell stage forming 8-cell stage 0.25 mm Animal pole Blastula (cross section) Vegetal pole Blastocoel Blastula (at least 128 cells). As cleavage continues, a fluid-filled cavity, the blastocoel, forms within the embryo. Because of unequal cell division due to the large amount of yolk in the vegetal hemisphere, the blastocoel is located in the animal hemisphere, as shown in the cross section. The SEM shows the outside of a blastula with about 4,000 cells, looking at the animal pole. Chapter 47: Animal Development 1. 2. 3. 4. 5. What exactly happens when sperm meets egg? What happens with activation of the egg? What happens in mammals? What happens with during cleavage? What is gastrulation? - Movement of blastula cells into the blastopore creating 2 cell (germ) layers - Ectoderm – outer layer - Endoderm – inner layer - Mesoderm – forms in between Figure 47.12 Gastrulation in a frog embryo 1 Gastrulation begins when a small indented crease, the dorsal lip of the blastopore, appears on one side of the blastula. The crease is formed by cells changing shape and pushing inward from the surface (invagination). Additional cells then roll inward over the dorsal lip (involution) and move into the interior, where they will form endoderm and mesoderm. Meanwhile, cells of the animal pole, the future ectoderm, change shape and begin spreading over the outer surface. SURFACE VIEW Animal pole CROSS SECTION Blastocoel Dorsal lip Dorsal lip Vegetal pole of blastopore Blastula of blastopore Blastocoel shrinking 2 The blastopore lip grows on both sides of the embryo, as more cells invaginate. When the sides of the lip meet, the blastopore forms a circle that becomes smaller as ectoderm spreads downward over the surface. Internally, continued involution expands the endoderm and mesoderm, and the archenteron begins to form; as a result, the blastocoel becomes smaller. 3 Late in gastrulation, the endoderm-lined archenteron has completely replaced the blastocoel and the three germ layers are in place. The circular blastopore surrounds a plug of yolk-filled cells. Blastocoel remnant Archenteron Ectoderm Mesoderm Endoderm Key Future ectoderm Future mesoderm Future endoderm Yolk plug Yolk plug Gastrula Chapter 47: Animal Development 1. 2. 3. 4. 5. 6. What exactly happens when sperm meets egg? What happens with activation of the egg? What happens in mammals? What happens with during cleavage? What is gastrulation? What is organogenesis? - Creation of organs - Involves folds, splits & clustering of cells - 1st organs are neural tube & notocord Chapter 47: Animal Development Students Correlations available now – sorry for the delay Learning Log – later today AP checks?? – March 9 deadline Has anyone not taken the Biology EOC? Transfers, movers, Figure 47.14 Early organogenesis in a frog embryo Neural folds Eye Neural fold Tail bud Neural plate SEM LM Somites Neural tube 1 mm 1 mm Neural fold Notochord Neural plate Neural crest Coelom Neural crest Somite Notochord Ectoderm Mesoderm Outer layer of ectoderm Endoderm Archenteron Neural crest (a) Neural plate formation. By the time shown here, the notochord has developed from dorsal mesoderm, and the dorsal ectoderm has thickened, forming the neural plate, in response to signals from the notochord. The neural folds are the two ridges that form the lateral edges of the neural plate. These are visible in the light micrograph of a whole embryo. Archenteron (digestive cavity) Neural tube (b) Formation of the neural tube. Infolding and pinching off of the neural plate generates the neural tube. Note the neural crest cells, which will migrate and give rise to numerous structures. (c) Somites. The drawing shows an embryo after completion of the neural tube. By this time, the lateral mesoderm has begun to separate into the two tissue layers that line the coelom; the somites, formed from mesoderm, flank the notochord. In the scanning electron micrograph, a side view of a whole embryo at the tail-bud stage, part of the ectoderm has been removed, revealing the somites, which will give rise to segmental structures such as vertebrae and skeletal muscle. Figure 47.16 Adult derivatives of the three embryonic germ layers in vertebrates ECTODERM • Epidermis of skin and its derivatives (including sweat glands, hair follicles) • Epithelial lining of mouth and rectum • Sense receptors in epidermis • Cornea and lens of eye • Nervous system • Adrenal medulla • Tooth enamel • Epithelium or pineal and pituitary glands MESODERM • Notochord • Skeletal system • Muscular system • Muscular layer of stomach, intestine, etc. • Excretory system • Circulatory and lymphatic systems • Reproductive system (except germ cells) • Dermis of skin • Lining of body cavity • Adrenal cortex ENDODERM • Epithelial lining of digestive tract • Epithelial lining of respiratory system • Lining of urethra, urinary bladder, and reproductive system • Liver • Pancreas • Thymus • Thyroid and parathyroid glands Chapter 47: Animal Development 1. 2. 3. 4. 5. 6. 7. What exactly happens when sperm meets egg? What happens with activation of the egg? What happens in mammals? What happens with during cleavage? What is gastrulation? What is organogenesis? What are the 4 extra-embryonic membranes in the amniotic egg? - Amnion - Allantois - Chorion - Yolk sac Figure 47.17 Extraembryonic membranes in birds and other reptiles Amnion. The amnion protects the embryo in a fluid-filled cavity that prevents dehydration and cushions mechanical shock. Allantois. The allantois functions as a disposal sac for certain metabolic wastes produced by the embryo. The membrane of the allantois also functions with the chorion as a respiratory organ. Embryo Albumen Amniotic cavity with amniotic fluid Shell Chorion. The chorion and the membrane of the allantois exchange gases between the embryo and the surrounding air. Oxygen and carbon dioxide diffuse freely across the egg’s shell. Yolk (nutrients) Yolk sac. The yolk sac expands over the yolk, a stockpile of nutrients stored in the egg. Blood vessels in the yolk sac membrane transport nutrients from the yolk into the embryo. Other nutrients are stored in the albumen (the ”egg white”). Chapter 47: Animal Development 1. 2. 3. 4. 5. 6. 7. 8. What exactly happens when sperm meets egg? What happens with activation of the egg? What happens in mammals? What happens with during cleavage? What is gastrulation? What is organogenesis? What are the 4 extra-embryonic membranes in the amniotic egg? How does mammalian development occur? - Slow cleavage - 1st division – 36 hrs - 2nd – 60 hrs - 3rd – 72 hrs Figure 47.18 Four stages in early embryonic development of a human Endometrium (uterine lining) Inner cell mass Trophoblast Blastocoel 1 Blastocyst reaches uterus. Maternal blood vessel Expanding region of trophoblast Epiblast Hypoblast Trophoblast 2 Blastocyst implants. Expanding region of trophoblast Amnion Amniotic cavity Epiblast Hypoblast 3 Extraembryonic membranes start to form and gastrulation begins. Chorion (from trophoblast) Extraembryonic mesoderm cells (from epiblast) Allantois Yolk sac (from hypoblast) Amnion Chorion Ectoderm Mesoderm Endoderm 4 Gastrulation has produced a threelayered embryo with four extraembryonic membranes. Yolk sac Extraembryonic mesoderm Chapter 47: Animal Development 1. 2. 3. 4. 5. 6. 7. 8. 9. What exactly happens when sperm meets egg? What happens with activation of the egg? What happens in mammals? What happens with during cleavage? What is gastrulation? What is organogenesis? What are the 4 extra-embryonic membranes in the amniotic egg? How does mammalian development occur? What three things influence cell fate? - Cytoplasmic determinants – mRNA & proteins in egg cytoplasm - Induction – cellular peer pressure - Cleavage pattern – divides cytoplasmic determinants Figure 21.11 Sources of developmental information for the early embryo Unfertilized egg cell Sperm Molecules of a a cytoplasmic determinant Molecules of another cytoplasmic determinant Fertilization Nucleus Zygote (fertilized egg) Mitotic cell division Two-celled embryo (a) Cytoplasmic determinants in the egg. The unfertilized egg cell has molecules in its cytoplasm, encoded by the mother’s genes, that influence development. Many of these cytoplasmic determinants, like the two shown here, are unevenly distributed in the egg. After fertilization and mitotic division, the cell nuclei of the embryo are exposed to different sets of cytoplasmic determinants and, as a result, express different genes. Figure 21.11b Early embryo (32 cells) NUCLEUS Signal transduction pathway Signal receptor Signal molecule (inducer) (b) Induction by nearby cells. The cells at the bottom of the early embryo depicted here are releasing chemicals that signal nearby cells to change their gene expression. Cellular peer pressure Figure 47.24 How does distribution of the gray crescent at the first cleavage affect the potency of the two daughter cells? EXPERIMENT 1 Gray crescent Left (control): Fertilized salamander eggs were allowed to divide normally, resulting in the gray crescent being evenly divided between the two blastomeres. Right (experimental): Fertilized eggs were constricted by a thread so that the first cleavage plane restricted the gray crescent to one blastomere. Gray crescent 2 The two blastomeres were then separated and allowed to develop. Normal Belly piece Normal RESULTS Blastomeres that receive half or all of the gray crescent develop into normal embryos, but a blastomere that receives none of the gray crescent gives rise to an abnormal embryo without dorsal structures. Spemann called it a “belly piece.” CONCLUSION The totipotency of the two blastomeres normally formed during the first cleavage division depends on cytoplasmic determinants localized in the gray crescent. Figure 47.25 Can the dorsal lip of the blastopore induce cells in another part of the amphibian embryo to change their developmental fate? EXPERIMENT Spemann and Mangold transplanted a piece of the dorsal lip of a pigmented newt gastrula to the ventral side of the early gastrula of a nonpigmented newt. Pigmented gastrula (donor embryo) Dorsal lip of blastopore Nonpigmented gastrula (recipient embryo) RESULTS During subsequent development, the recipient embryo formed a second notochord and neural tube in the region of the transplant, and eventually most of a second embryo. Examination of the interior of the double embryo revealed that the secondary structures were formed in part from host tissue. Primary embryo Secondary structures: Notochord (pigmented cells) Neural tube (mostly nonpigmented cells) Primary structures: Secondary (induced) embryo Neural tube Notochord CONCLUSION The transplanted dorsal lip was able to induce cells in a different region of the recipient to form structures different from their normal fate. In effect, the dorsal lip “organized” the later development of an entire embryo. Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? 4. What happens with during cleavage? 5. What is gastrulation? 6. What is organogenesis? 7. What are the 4 extra-embryonic membranes in the amniotic egg? 8. How does mammalian development occur? 9. What three things influence cell fate? 10. How are organisms formed from the fertilized egg? - Cell division/cleavage - Morphogenesis – process of giving shape to an organism - Cell differentiation – process by which cells become specialized Figure 21.4 Some key stages of development in animals and plants (a) Animal development. Most animals go through some variation of the blastula and gastrula stages. The blastula is a sphere of cells surrounding a fluid-filled cavity. The gastrula forms when a region of the blastula folds inward, creating a tube—a rudimentary gut. Once the animal is mature, differentiation occurs in only a limited way—for the replacement of damaged or lost cells. Cell movement Zygote (fertilized egg) Eight cells Blastula (cross section) Gut Gastrula Adult animal (cross section) (sea star) Cell division Morphogenesis (b) Plant development. In plants with seeds, a complete embryo develops within the seed. Morphogenesis, which involves cell division and cell wall expansion rather than cell or tissue movement, occurs throughout the plant’s lifetime. Apical meristems (purple) continuously arise and develop into the various plant organs as the plant grows to an indeterminate size. Observable cell differentiation Seed leaves Shoot apical meristem Zygote (fertilized egg) Root apical meristem Two cells Embryo inside seed Plant Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? 4. What happens with during cleavage? 5. What is gastrulation? 6. What is organogenesis? 7. What are the 4 extra-embryonic membranes in the amniotic egg? 8. How does mammalian development occur? 9. What three things influence cell fate? 10. How are organisms formed from the fertilized egg? 11. Can cells de-differentiate? - Plant cuttings - Animal cells???? Figure 21.6 Can the nucleus from a differentiated animal cell direct development of an organism? EXPERIMENT Researchers enucleated frog egg cells by exposing them to ultraviolet light, which destroyed the nucleus. Nuclei from cells of embryos up to the tadpole stage were transplanted into the enucleated egg cells. Frog embryo Frog egg cell Fully differentiated (intestinal) cell Less differentiated cell Donor nucleus transplanted Most develop into tadpoles Frog tadpole Enucleated egg cell Donor nucleus transplanted <2% develop into tadpoles Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? 4. What happens with during cleavage? 5. What is gastrulation? 6. What is organogenesis? 7. What are the 4 extra-embryonic membranes in the amniotic egg? 8. How does mammalian development occur? 9. What three things influence cell fate? 10. How are organisms formed from the fertilized egg? 11. Can cells de-differentiate? 12. How was Dolly cloned? - Nuclear transplantation Fig. 21.7 Reproductive Cloning of a Mammal by Nuclear Transplantation APPLICATION This method is used to produce cloned animals whose nuclear genes are identical to the donor animal supplying the nucleus. 1 RESULTS The cloned animal is identical in appearance and genetic makeup to the donor animal supplying the nucleus, but differs from the egg cell donor and surrogate mother. 2 Egg cell from ovary Nucleus Nucleus removed 3 Cells fused removed TECHNIQUE Shown here is the procedure used to produce Dolly, the first reported case of a mammal cloned using the nucleus of a differentiated cell. Egg cell donor Mammary cell donor Cultured mammary cells are semistarved, arresting the cell cycle and causing dedifferentiation Nucleus from mammary cell 4 Grown in culture Early embryo 5 Implanted in uterus of a third sheep 6 Embryonic development Surrogate mother Lamb (“Dolly”) genetically identical to mammary cell donor Figure 21.9 Working with stem cells Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equivalent of blastula) From bone marrow in this example Totipotent cells Pluripotent cells Cultured stem cells Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? 4. What happens with during cleavage? 5. What is gastrulation? 6. What is organogenesis? 7. What are the 4 extra-embryonic membranes in the amniotic egg? 8. How does mammalian development occur? 9. What three things influence cell fate? 10. How are organisms formed from the fertilized egg? 11. Can cells de-differentiate? 12. How was Dolly cloned? 13. When is a cell determined (fated)? - Muscle cells – MyoD transcription factor – turns on all muscle genes Figure 21.10 Determination and differentiation of muscle cells Nucleus Master control gene myoD Other muscle-specific genes DNA Embryonic precursor cell OFF OFF Figure 21.10 Determination and differentiation of muscle cells Nucleus Master control gene myoD Other muscle-specific genes DNA OFF Embryonic precursor cell 1 Myoblast (determined) Determination. Signals from other cells lead to activation of a master regulatory gene called myoD, and the cell makes MyoD protein, a transcription factor. The cell, now called a myoblast, is irreversibly committed to becoming a skeletal muscle cell. OFF OFF mRNA MyoD protein (transcription factor) Figure 21.10 Determination and differentiation of muscle cells Nucleus Master control gene myoD Other muscle-specific genes DNA OFF Embryonic precursor cell 1 Myoblast (determined) 2 Determination. Signals from other cells lead to activation of a master regulatory gene called myoD, and the cell makes MyoD protein, a transcription factor. The cell, now called a myoblast, is irreversibly committed to becoming a skeletal muscle cell. Differentiation. MyoD protein stimulates the myoD gene further, and activates genes encoding other muscle-specific transcription factors, which in turn activate genes for muscle proteins. MyoD also turns on genes that block the cell cycle, thus stopping cell division. The nondividing myoblasts fuse to become mature multinucleate muscle cells, also called muscle fibers. OFF OFF mRNA MyoD protein (transcription factor) mRNA MyoD Muscle cell (fully differentiated) mRNA Another transcription factor mRNA mRNA Myosin, other muscle proteins, and cell-cycle blocking proteins Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? 4. What happens with during cleavage? 5. What is gastrulation? 6. What is organogenesis? 7. What are the 4 extra-embryonic membranes in the amniotic egg? 8. How does mammalian development occur? 9. What three things influence cell fate? 10. How are organisms formed from the fertilized egg? 11. Can cells de-differentiate? 12. How was Dolly cloned? 13. When is a cell determined (fated)? 14. What is apoptosis? - Programmed cell death – cell suicide Figure 21.18 Molecular basis of apoptosis in C. elegans Ced-9 protein (active) inhibits Ced-4 activity Death signal receptor Mitochondrion Ced-4 Ced-3 Inactive proteins Cell forms blebs (a) No death signal Ced-9 (inactive) Death signal Active Active Ced-4 Ced-3 Activation cascade (b) Death signal Other proteases Nucleases Chapter 47: Animal Development 1. What exactly happens when sperm meets egg? 2. What happens with activation of the egg? 3. What happens in mammals? 4. What happens with during cleavage? 5. What is gastrulation? 6. What is organogenesis? 7. What are the 4 extra-embryonic membranes in the amniotic egg? 8. How does mammalian development occur? 9. What three things influence cell fate? 10. How are organisms formed from the fertilized egg? 11. Can cells de-differentiate? 12. How was Dolly cloned? 13. When is a cell determined (fated)? 14. What is apoptosis? 15. What are some model organisms for studying development? Figure 21.2 Model Organisms for Genetic Studies of Development DROSOPHILA MELANOGASTER (FRUIT FLY) Drosophila - small, easy & cheap to culture - 2 week generation time - 4 chromosomes - LARGE literature of info CAENORHABDITIS ELEGANS (NEMATODE) C elegans - easy to culture - transparent body with few cell types - zygote to mature adult in 3 days 0.25 mm ARABIDOPSIS THAMANA (COMMON WALL CRESS) MUS MUSCULUS (MOUSE) Mouse - vertebrate - LARGE literature - transgenics & knock-outs DANIO RERIO (ZEBRAFISH)