LECTURE PRESENTATIONS For CAMPBELL BIOLOGY NINTH EDITION Jane

LECTURE PRESENTATIONS For CAMPBELL BIOLOGY, NINTH EDITION Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson Chapter 47 Animal Development Lectures by Erin Barley Kathleen Fitzpatrick © 2011 Pearson Education, Inc.

Overview: A Body-Building Plan • A human embryo at about 7 weeks after conception shows development of distinctive features Eye Heart Vertebrae

• Development occurs at many points in the life cycle of an animal • This includes metamorphosis and gamete production, as well as embryonic development EMBRYONIC DEVELOPMENT TIO FER TIL Egg Zygote IZA Adult frog N Sperm Metamorphosis LE GCA ST RU LAT NO GA IS OR NES GE Larval stages E AG AV Tail-bud embryo ION Blastula Gastrula

• Although animals display different body plans, they share many basic mechanisms of development and use a common set of regulatory genes • Biologists use model organisms to study development, chosen for the ease with which they can be studied in the laboratory

Fertilization • Fertilization is the start of embryonic development – the formation of a diploid zygote from a haploid egg and sperm – takes place in the first third of the human fallopian tube • two types in animals • 1. Internal – mammals, reptiles, amphibians, worms • 2. External – echinoderms, cnidarians, fish • molecules and events at the egg surface play a crucial role in each step of fertilization – sperm penetrate the protective layer around the egg – receptors on the egg surface bind to molecules on the sperm surface – changes at the egg surface prevent polyspermy = the entry of multiple sperm nuclei into the egg

The Acrosomal Reaction • • first studied with sea urchin eggs any similarities with mammals the acrosomal reaction is triggered when the sperm meets the egg binding of the sperm to a receptor on the egg triggers this reaction – docking onto the vitelline layer activates the acrosome at the tip of the sperm – acrosome releases hydrolytic enzymes that digest the coating surrounding the egg – the sperm extends an acrosomal process through the VL – process docks with a receptor on the egg depolarization of the egg and fast block to polyspermy Sperm plasma membrane Sperm nucleus Basal body (centriole) Sperm head Acrosome Jelly coat Sperm-binding Acrosomal process Fertilization envelope Actin filament Cortical Fused plasma granule membranes Hydrolytic enzymes Perivitelline Vitelline layer space EGG CYTOPLASM Egg plasma membrane

The Cortical Reaction • depolarization/fast block does not last very long • IN ADDITION: the sperm binding to receptors on the egg’s plasma membrane results in release of intracellular calcium • calcium release stimulates exocytosis from cortical granules – Not sure about the contents of these granules • granule contents build up in between the egg’s membrane and the VL - do two things: – 1. removes the sperm receptors from the plasma membrane – 2. hardens the vitelline layer and forms a fertilization envelope • VL is now impervious to more sperm = slow block to polyspermy Sperm plasma membrane Sperm nucleus Basal body (centriole) Sperm head Acrosome Jelly coat Sperm-binding receptors Acrosomal process Fertilization envelope Actin filament Cortical Fused granule plasma membranes Hydrolytic enzymes Perivitelline space Vitelline layer EGG CYTOPLASM Egg plasma membrane

• the continued build up of these granules draws water into the space between the plasma membrane of the egg and the fertilization envelope – lifts is away and “rips” all other sperm off the egg – except for the one that is attached by its acrosomal process • followed by the entry of the sperm’s nucleus • eventual fusion of the sperm and egg nuclei • BUT – the egg must be activated first Sperm plasma membrane Sperm nucleus Basal body (centriole) Sperm head Acrosome Jelly coat Sperm-binding receptors Fertilization envelope Acrosomal process Actin filament Cortical Fused granule plasma membranes Hydrolytic enzymes Perivitelline space Vitelline layer Egg plasma membrane EGG CYTOPLASM

Fertilization in Mammals • fertilization in mammals and other terrestrial animals is internal • secretions in the mammalian female reproductive tract alter sperm motility and structure – this is called capacitation – must occur before sperm are able to fertilize an egg • plasma membrane of the egg is surrounded by an extracellular matrix = zona pellucida – similar to the vitelline layer • and a ring of follicular cells = corona radiata (nourishment)

1. several sperm enter zona pellucida -one of the glycoproteins within the ZP (ZP 3) acts as a receptor for the sperm -initiates an acrosomal reaction: -production of hyaluornidase to digest away the corona radiata -production of acrosin to digest away the ZP and oocyte membrane -exposes a protein on the sperm (Gal. T) that allows it to bind to the egg’s plasma membrane 2. the first sperm to contact the plasma membrane of the egg triggers the slow block to polyspermy 3. oocyte releases the hardened zona pellucida away from the egg surface 4. entry of the entire sperm into the egg’s cytoplasm 5. fusion of the sperm with nucleus of the egg -before entry and fusion by the sperm - the secondary oocyte must complete meiosis II and form the ovum Zona pellucida • No fast block to polyspermy has been identified in mammals Follicle cell Sperm Cortical Sperm nucleus granules basal body

Egg Activation • fusion of the two nuclei requires egg activation • the rise in Ca 2+ in the cytosol of the egg increases the rates of cellular respiration and protein synthesis by the egg cell – when this happens - egg is said to be activated • the proteins and m. RNAs needed for activation are already present in the egg • one key event in oocyte activation = completion of meiosis II to form the ovum -triggered by calcium release inside cell • following activation the sperm nucleus can merge with the egg nucleus and cell division begins

Gamete fusion in Mammals -sperm and egg membranes fuse entire sperm taken into the egg -about 4 hours later – nuclei fusion -the nuclear envelopes of the egg and sperm disappear -sperm and egg chromosomes organized onto the same mitotic spindle -after this = diploid nucleus and a zygote – in mammals - the first cell division occurs 12 36 hours after sperm binding Zona pellucida -the sperm contributes its genome and one centriole -other organelles of the sperm are rapidly degraded – including its mitochondria which are thought to be mutated because of the stress of “swimming” to the oocyte (higher metabolism causes stress damage!) Follicle cell Sperm Cortical Sperm nucleus granules basal body

The One-celled zygote • fertilized egg = zygote • Greek for “to join” or “to yolk” • comprised of: – diploid nucleus – cytoplasm – contains yolk that is taken up during oogenesis via endocytosis • mitosis and cytokinesis of the one-celled zygote will duplicate the nucleus and partition the cytoplasm into the progeny cells – known as Cleavage – cleavage partitions the cytoplasm of the zygote (one large cell) into many smaller cells called blastomeres – division of the one celled-zygote creates the embryo two nuclei fusing to form the zygote

What is Yolk? • • food source stored in the egg’s cytoplasm for growth of the embryo made up of yolk proteins and lipids amount of yolk differs from animal to animal – fish, amphibians, birds and some insects have “yolky” eggs – mammals – small oocytes with very little yolk • most yolk is not made by the oocyte – made by the liver and are transported by the circulatory system of the animal to the forming oocyte • the yolk is heavy and is found at the bottom of the oocyte = site of the vegetal pole or vegetal hemisphere • in mammals – the yolk is distributed throughout the oocyte Yolk proteins

The one-celled Zygote • the zygote is a single cell with ONE nucleus (containing 46 single chromatid chromosomes) plus a cytoplasm containing yolk proteins and lipids (i. e. yolk) • in zygotes with large amounts of yolk: – because the yolk is heavy it settles to the bottom of the zygote (i. e. vegetal pole) – this “squishes” the nucleus up toward the opposite pole = animal pole – as the zygote splits and forms the embryo – it forms on top of the yolk • in zygotes with little yolk – not seen

The one-celled Zygote • as the zygote splits and forms the embryo – cytokinesis will partition the yolk into the blastomeres of the embryo – those with the most yolk stay at the vegetal pole of the embryo

The One-celled zygote • the difference in yolk distribution as cleavage proceeds results in animal and vegetal hemispheres that differ in appearance • two poles in the zygote – animal and vegetal with a marginal zone in between called a gray crescent • animal pole – region of the zygote where blastomere division is rapid – little yolk within these cells – also the location of sperm entry – cells will become the embryo’s ectoderm and endoderm • vegetal pole - has more yolk in its blastomeres – – cells are larger divide slower some of the cells will become the embryo’s endoderm in some animals – also forms the extra-embryonic membranes Yolk proteins

Cleavage • zygote undergoes cleavage - a period of rapid cell division without growth – cell cycle consists mainly of the S phase and M phase (DNA synthesis and mitosis) – very little protein synthesis done – skips the G 1 and G 2 phases • first 5 to 7 divisions produces the blastula – a hollow ball of cells with a fluid-filled cavity called a blastocoel 50 m (a) Fertilized egg (b) Four-cell stage (c) Early blastula (d) Later blastula

Cleavage Patterns • cleavage patterns studied in the frog Xenopus • in frogs and many other animals- the distribution of yolk is a key factor influencing the pattern of cleavage • types of cleavage: – 1. Determinate: results in the developmental fate of the cells within an embryo being set very early on • e. g. 2 cell stage – 2. Indeterminate: fate is not established until later on in development • cleavage patterns: – A. Holoblastic – B. Meroblastic

• Holoblastic cleavage = complete division of the egg – occurs in species whose eggs have little or moderate amounts of yolk – e. g. sea urchins and frogs and mammals – types: • • 1. bilateral 2. radial 3. rotational 4. spiral • Meroblastic cleavage = incomplete division of the egg – occurs in species with yolk-rich eggs – the cleavage furrow cannot pass completely through the cell – e. g. reptiles and birds – types: • 1. discoidal • 2. superficial – mitosis with no cytokinesis Worms Humans Frog Radial Spiral

Cleavage Pattern in the Frog • 2 cell stage: first cleavage furrow extends from the animal to the vegetal pole – cuts through the gray crescent – establishes the anterior-posterior axis of the animal • 4 cell stage: second cleavage furrows also extends from the animal to the vegetal pole – forms four equally sized blastomeres Zygote 2 -cell stage forming Gray crescent 0. 25 mm 4 -cell stage forming Animal pole 8 -cell stage (viewed from the animal pole) 0. 25 mm Vegetal pole Blastocoel Blastula (cross section) Blastula (at least 128 cells)

Cleavage Pattern in the Frog • 8 cell stage: the third cleavage is asymmetric and is along the “equator” between the two poles – BUT: the amount of yolk in the cells of the vegetal pole displaces the cleavage furrow toward the animal pole – forms unequally sized blastomeres – animal pole blastomeres are smaller in size (micromeres) • this displacing effect continues from this point -more cells found at the animal pole • 16 -cell stage: known as the morula • rearrangement of the cells of the morula results in the blastula • cleavage is considered done as the embryo transitions from morula to blastula Zygote 2 -cell stage forming Gray crescent 0. 25 mm 4 -cell stage forming Animal pole 8 -cell stage (viewed from the animal pole) 0. 25 mm Vegetal pole Blastocoel Blastula (cross section) Blastula (at least 128 cells)

Cleavage Patterns in Amniotes • in amniotes - rearrangement of the cells of the morula results in a mass of cells at one end of the blastula – mass of cells = inner cell mass – fluid-filled cavity = blastocoel – outer covering = trophoblast Blastula

The Human Blastocyst -human embryonic development: -displacement of cleavage furrow doesn’t happen in mammals like humans – very little yolk in these cells -so cleavage pattern produces equal sized blastomeres -day 4– formation of 16 celled morula -day 5 -fluid begins to collect in the morula and reorganizes the cells around a blastocoel -embryo is now called a blastocyst (7 cleavages or ~130 cells) -outer layer = trophoblast -epithelial layer that forms extra-embryonic tissues (e. g. placenta, yolk sac) -also plays a role in implantation by breaking down the endometrium through the secretion of enzymes -inner cell mass at one end - totipotent embryonic stem cells

Blastula vs. Blastocyst • Blastula – hollow ball of cells (blastoderm) surrounding a fluidfilled cavity called a blastocoel – e. g. sea urchins • Blastula – hollow ball of cells (trophoblast) surrounding an inner cell mass at one end a blastocoel at the other – found in amniotes – animals that make an egg with an amniotic cavity • e. g. frogs, chickens • Blastocyst – blastula found in mammals – e. g. humans

Morphogenesis in animals involves specific changes in cell shape, position, and survival • after cleavage, the rate of cell division slows and the normal cell cycle is restored • Morphogenesis = the process by which cells occupy their appropriate locations and the embryo takes on its shape – occurs through the production of soluble factors that control the differentiation of cells = morphogens – form gradients within the embryo – many act as transcription factors and bind gene promoters – others control cell migration and cell-cell adhesion • morphogenesis involves: – 1. Gastrulation = the movement of cells from the blastula surface to the interior of the embryo – 2. Organogenesis = the formation of organs

Gastrulation • Gastrulation rearranges the cells of a blastula into a three-layered embryo = called a gastrula • the three layers produced by gastrulation are called embryonic germ layers – the ectoderm forms the outer layer – the endoderm lines the digestive tract – the mesoderm partly fills the space between the endoderm and ectoderm • Each germ layer contributes to specific structures in the adult animal

ECTODERM (outer layer of embryo) • Epidermis of skin and its derivatives (including sweat glands, hair follicles) • Nervous and sensory systems • Pituitary gland, adrenal medulla • Jaws and teeth • Germ cells MESODERM (middle layer of embryo) • Skeletal and muscular systems • Circulatory and lymphatic systems • Excretory and reproductive systems (except germ cells) • Dermis of skin • Adrenal cortex ENDODERM (inner layer of embryo) • Epithelial lining of digestive tract and associated organs (liver, pancreas) • Epithelial lining of respiratory, excretory, and reproductive tracts and ducts • Thymus, thyroid, and parathyroid glands

Gastrulation Definitions • invagination – infolding of a cell sheet into an embryo – forms the mouth, anus and archenteron • involution – turning in of a cell sheet • blastopore – opening of the archenteron – forms at the point where cells enter the embryo • deuterostome – blastopore becomes the anus • protostome – blastopore becomes the mouth

Gastrulation in Sea Urchins • • gastrulation begins at the vegetal pole of the blastula cells known as mesenchyme cells (red cells) detach and migrate throughout the blastocoel the remaining cells (yellow cells) near the vegetal plate flatten and buckle inward through a process called invagination the depression becomes bigger and deeper and becomes a cavity called the archenteron – – – • lined with endodermal cells will become the digestive tract the opening of the archenteron is the blastopore which will become the anus mesenchymal cells at the tip of the archenteron form projections called filopodia – – – Blastocoel Mesenchyme cells Vegetal plate Vegetal pole Blastocoel Filopodia Mesenchyme cells filopodia “drag” the archenteron through the blastocoel fuses with the wall of the blastocoel that opening becomes the mouth • since the mouth forms second the sea urchin is known as a Deuterostome Animal pole Key Future ectoderm Future mesoderm Future endoderm Blastopore Archenteron 50 m Blastocoel Ectoderm Mouth Mesenchyme (mesoderm forms future skeleton) Archenteron Blastopore Digestive tube (endoderm) Anus (from blastopore)

Video: Sea Urchin Embryonic Development © 2011 Pearson Education, Inc.

Gastrulation in Frogs • • begins when a group of cells on the dorsal side of the blastula begins to change shape and invaginate invagination forms a crease – – • • these cells will form the mesoderm and endoderm mesoderm stays at the periphery of the embryo the endoderm fills the embryo as more cells invaginate - the crease/blastopore expands and extends around the embryo (red arrows) the ectoderm expands and reduces the blastopore to a small area at the vegetal pole cells continue to enter into the embryo and the mesoderm and endoderm expands and forms the archenteron – • will form the blastopore the part above the crease is called the dorsal lip of the blastopore cells move into the interior (involution - dashed arrow) – – – 1 CROSS SECTION SURFACE VIEW Animal pole Blastocoel Dorsal lip of blastopore Early Vegetal pole gastrula Blastopore Blastocoel shrinking 2 3 Blastocoel remnant the blastocoel shrinks and eventually disappears Dorsal lip of blastopore Archenteron Ectoderm Mesoderm Endoderm late in gastrulation – blastopore forms the anus Key Future ectoderm Future mesoderm Future endoderm Blastopore Late gastrula Blastopore Yolk plug Archenteron

Gastrulation in Chicks • prior to gastrulation – the chick embryo is composed of an upper and lower layer that form an embryonic disk Fertilized egg Primitive streak Embryo – the epiblast and hypoblast – epiblast = embryo – hypoblast (‘roof’ of the yolk sac) = supports embryology & forms part of the yolk sac Yolk Primitive streak Epiblast Future ectoderm Blastocoel Migrating cells (mesoderm) Endoderm Hypoblast YOLK

Gastrulation in Chicks • during gastrulation - epiblast cells move toward the midline of the blastoderm • pile-up of cells at the midline forms a thickening called the primitive streak • epiblast cells migrate through the primitive streak toward the yolk Fertilized egg Primitive streak Embryo Yolk Primitive streak – some cells push the hypoblast to the side to become the endoderm – those remaining cells Future mesoderm ectoderm – leftover epiblast cells ectoderm • animals with three germ layers are known as Triploblastic • Gastrulation is marked by increased transcription and translation Epiblast Blastocoel Migrating cells (mesoderm) Endoderm Hypoblast YOLK

Gastrulation in Humans Endometrial epithelium (uterine lining) 1 Blastocyst reaches uterus. • human eggs have very little yolk Uterus – but we do have a yolk sac (site of hematopoeisis) • • • the blastocyst is the human equivalent of the blastula early stages are very similar to chick gastrulation end of day 5 – blastocyst squeezes out of the zona pellucida and is ready for implantation (day 7) – • • • trophoblast is critical for implantation following implantation - the trophoblast continues to expand forms the extraembryonic membranes Day 13 - gastrulation follows – similar to the chick embryo the front of the primitive streak forms the blastopore Inner cell mass Trophoblast Blastocoel 2 Blastocyst implants (7 days after fertilization). Expanding region of trophoblast Maternal blood vessel Epiblast Hypoblast Trophoblast 3 Extraembryonic membranes start to form (10– 11 days), and gastrulation begins (13 days). Expanding region of trophoblast Amniotic cavity Epiblast Hypoblast Yolk sac (from hypoblast) Extraembryonic mesoderm cells (from epiblast) Chorion (from trophoblast) 4 Gastrulation has produced a three-layered embryo with four extraembryonic membranes. Amnion Chorion Ectoderm Mesoderm Endoderm Yolk sac Extraembryonic mesoderm Allantois

Developmental Adaptations of Amniotes • colonization of land by vertebrates was made possible only after the evolution of two things: – 1. shelled egg of birds and other reptiles as well as monotremes (egglaying mammals) OR – 2. uterus of marsupial and eutherian mammals • in both - embryos are surrounded by fluid in a sac called the amnion – protects the embryo from desiccation and allows reproduction on dry land • mammals and reptiles including birds are called amniotes for this reason

• four extraembryonic membranes that form around the embryo – 1. chorion - functions in gas exchange • • • two layers – forms from the extraembryonic mesoderm and trophoblast projections enter into the endometrium = chorionic villi inner layer contacts the amnion – 2. amnion - encloses the amniotic fluid; protection of embryo • • from EE mesoderm and ectoderm early development not seen in humans sac forms just after formation of the blastocyst floor is the epiblast and then the ectoderm – 3. yolk sac - encloses the yolk in bony fishes, sharks, reptiles and birds • • • also exists in mammals humans – primitive circulatory system and liver roof is the hypoblast and then the endoderm – 4. allantois - disposes of waste products and contributes to gas exchange • • • not seen in fish and amphibians filled with blood vessels – O 2 transport storage site for nitrogenous wastes placental mammals – forms part of the umbilical cord humans – also a connection point to the fetal bladder

Organogenesis • during organogenesis - various regions of the germ layers develop into rudimentary organs • two important ones to a vertebrate: notochord and the neural plate – the notochord forms from mesoderm – the neural plate forms from ectoderm

-four weeks of development - embryo forms a tubular structure and undergoes Neurulation -embryo begins to form definitive structures – undergoes morphogenesis PLUS organogenesis: ** neurulation occurs by induction (one tissue influences the development of another) -e. g. formation of the nervous system requires the mesodermal cells of the notochord

-in front of the primitive streak - primitive node/Hensen’s knot secretion of numerous growth factors for neural development -in front of the primitive node - the ectoderm folds to form the neural folds head and associated structures -the groove between the folds (neural groove) deepens and the folds “fold over” to form a tube = neural tube -the neural groove develops into three ‘vesicles’ forebrain, midbrain and hindbrain -mesodermal cells of the primitive node form a tube that runs the length of the embryo - becomes the notochord (day 22 -24) vertebral column

• notochord: Neural folds – develops from the mesoderm of the embryo – supportive rod that extends most of the animal’s length – extends into the tail – dorsal to the body cavity • located between the nerve cord and the digestive tract – flexible to allow for bending but resists compression 1 mm Neural fold plate • also a point of swimming muscle attachment in some species – e. g. amphioxus – composed of large, fluid-filled cells encased in a fairly stiff fibrous tissue – will become the vertebral column in many chordates • in humans, remnants of the notochord can be found in the intervertebral discs Notochord Ectoderm Mesoderm Endoderm Archenteron (a) Neural plate formation

Video: Frog Embryo Development

• the ectoderm on top of the notochord is the neural plate • • requires the formation of the notochord first – induction by the mesoderm Neural fold Neural plate the neural plate curves inward bringing the neural folds together and forming the neural tube (Neurulation) – tissue on top of the neural tube is ectoderm and will form the outer covering of the animal • the neural tube will become the central nervous system (brain and spinal cord) – will form into a dorsal nerve cord (spinal cord) – anterior expansion are called neural folds and will form the brain • • cells that do not form the neural plate or stay in the ectoderm are called Neural crest cells neural crest cells migrate throughout the embryo to form many tissues – nerves, parts of teeth, skull bones, part of the heart (b) Neural tube formation Neural crest cells Neural tube Outer layer of ectoderm

• portions of the mesoderm that do not form the notochord form blocks called somites – located lateral to the notochord – arranged serially along the length of the notochord – differentiate into sclerotomes (cartilage and tendons, vertebrae), myotomes (skeletal muscle) and dermatomes (dermis) – other parts differentiate into mesenchymal cells - migratory • in vertebrates - one of the major functions of the somites is to form the vertebrae, muscles of the vertebrae and the ribs • lateral to the somites - the mesoderm splits into two layers Eye SEM Neural tube Notochord Coelom Somites Tail bud 1 mm Neural crest cells Somite – defines the coelom (body cavity) (c) Somites Archenteron (digestive cavity)

• Organogenesis in the chick is quite similar to that in the frog Neural tube Notochord Eye Forebrain Somite Coelom Endoderm Mesoderm Ectoderm Archenteron Lateral fold Heart Blood vessels Somites Yolk stalk These layers form extraembryonic membranes. (a) Early organogenesis Yolk sac Neural tube YOLK (b) Late organogenesis

• The mechanisms of organogenesis in invertebrates are similar – but the body plan is very different – e. g. the neural tube develops along the ventral side of the embryo in invertebrates, rather than dorsally as occurs in vertebrates

Mechanisms of Morphogenesis • morphogenesis = creation of form or shape • morphogenesis in animals but not plants involves movement of cells – cell wall of plants prevents complex processes like gastrulation and organogenesis • movement in parts of the cell – changes in the cytoskeleton can result in cell shape changes • movement of the cell itself = migration

The Cytoskeleton in Morphogenesis • reorganization of the cytoskeleton is a major force in changing cell shape during development • e. g. neurulation - microtubules oriented from dorsal to ventral in a sheet of ectodermal cells help lengthen the cells along that axis and creates a long, rolled tubular embryo • 1. cuboidal ectodermal cells form a continuous sheet • 2. microtubules elongate the cells • 3. actin filaments contract and deform the sheet into a wedge – invagination results • 4. a wedge at the bottom and at the top creates a tube • 5. pinching off forms the tube Ectoderm Neural plate Microtubules Actin filaments Neural tube

• the cytoskeleton also directs cell migration – during organogenesis • cells “crawl” during embryonic development – cytoskeleton produces cellular extensions that adhere to substrates and retracts • the production of an extracellular matrix within the embryo can direct where these cells will go – production of cellular adhesion molecules (CAMs) on the surface of the migrating cells interact with the ECM – these CAMs interact with the cytoskeleton • production of growth factor gradients can also direct migration

Programmed Cell Death • also known as apoptosis • at various times during development - individual cells, sets of cells, or whole tissues stop developing and undergo apoptosis – are engulfed by neighboring cells • e. g. loss of the tail by the tadpole frog • e. g. loss of the webbing between fingers and toes humans • e. g. many more neurons are produced in developing embryos than will be needed – extra neurons are removed by apoptosis • numerous pathways regulate apoptosis – production and activation of proteins called caspases – also a role for the mitochondria in triggering the activation of these caspases

Cytoplasmic determinants and inductive signals contribute to cell fate specification • Cells in a multicellular organism share the same genome – • how does one cell differ from another? ? Differences in cell types are the result of the expression of different sets of genes – cell is said to have acquired a specific fate • there are two ways cell fate can be determined – 1. Irreverisibly Determination – 2. Reversibly Specification or Differentiation • Determination is the term used to describe the process by which a cell or group of cells becomes permanently committed to a particular fate – e. g. dorsal vs. ventral structures – occur VERY early on in embryonic development – determination cannot be reversed! • Specification or Differentiation refers to the resulting specialization in structure and function – this can be reversed

Cytoplasmic determinants and inductive signals contribute to cell fate specification • determination and specification can be done of two ways: – autonomous is controlled by cytoplasmic determinants • no role for its surrounding environment • factors made within the cytoplasm • usually are transcription factors that alter gene expression – conditional is controlled by extracellular factors such as surrounding cells or growth factors

Cytoplasmic determinants and inductive signals contribute to cell fate specification • fate determination and differentiation is a series of complex events that involve changes in gene expression – inductive signals for these changes are also complex – can involve many things – such as: • production of growth factors within the embryo – triggers specific signaling pathways within the target cell changes in gene expression • production of the extracellular matrix – can affect signaling pathways within the cell also changes in gene expression • physical interaction between two cells

Determination and Specification • the sea squirt (tunicate) and conditional specification: – transplantation of the part of the cytoplasm (yellow crescent) produces muscle cells – endodermal cells produce FGF notochord development by anteriorly positioned cells; mesenchyme by posteriorly positioned cells • the sea urchin: – anterior-posterior axis lies along the animal-vegetal axis – the blastomeres in the vegetal pole induce nearby tissue to become endoderm and only endoderm • cell fate determination • role for a protein called beta-catenin • present in the nuclei of cells in the vegetal pole – autonomous specification – the blastomeres of the animal pole induce nearby cells to become either ectoderm, mesoderm or endoderm • cell fate specification because those cells that choose ectoderm can reverse and become endoderm or mesoderm • autonomous and conditional specification

Fate Mapping • Fate maps are diagrams showing organs and other structures that arise from each region of an embryo • classic studies using frogs indicated that cell lineage in germ layers is traceable to blastula cells – 1920 s – Walther Vogt – marked specific cells in a frog blastula with non-toxic dyes – embryos sectioned and the dyes detected • also done in tunicates (primitive chordates) Epidermis Central nervous system Epidermis Notochord Mesoderm Endoderm Blastula Neural tube stage (transverse section) (a) Fate map of a frog embryo 64 -cell embryos Blastomeres injected with dye Larvae (b) Cell lineage analysis in a tunicate

• later studies of C. elegans used the ablation (destruction) of single cells to determine the structures that normally arise from each cell – Caenorhabditis elegans is a roundworm – 1 mm long – transparent body • researchers were able to determine the lineage of each of the 959 somatic cells in the worm Time after fertilization (hours) – determined that exactly 131 cells dies during normal development – if one gene in these cells is mutated – they all live – gene was found to be critical in apoptosis across a wide range of animals Zygote 0 First cell division Nervous system, outer skin, musculature 10 Musculature, gonads Outer skin, nervous system Germ line (future gametes) Musculature Hatching Intestine Mouth Anus Eggs Vulva 1. 2 mm ANTERIOR POSTERIOR

• cell fate mapping in C. elegans done using germ cells – are determined cells that give rise to sperm or eggs • in all animals - complexes of RNA and protein are involved in the specification of germ cell fate • in C. elegans - such complexes are called P granules – found in the zygote and persist throughout development – can be detected in the gonads of the adult worm – labelling of P granules in a fertilized C. elegans egg can allow researchers to track what happens as a germ cell’s fate is determined 100 m

• P granules are distributed throughout the newly fertilized C. elegans egg and move to the posterior end before the first cleavage division – as a result only the most posterior of the two cells formed by the first mitotic division contains P granules 20 m 1 Newly fertilized egg • with each subsequent cleavage, the P granules are partitioned into the posteriormost cells – this cell is now a germ cell 2 Zygote prior to first division • P granules act as cytoplasmic determinants - fixing germ cell fate at the earliest stage of development – fate is not reversible • tracking labelled P granules allows us to see where germ cells develop and migrate in an embryo 3 Two-cell embryo 4 Four-cell embryo

Axis Formation in the frog • a body plan with bilateral symmetry is found across a range of animals – nematodes, annelids, several invertebrates and also vertebrates – this body plan exhibits asymmetry across the dorsal-ventral and anterior-posterior axes – the right-left axis is largely symmetrical • the anterior-posterior axis of the frog embryo is determined during oogenesis – cleavage along the animal-vegetal pole indicates where the anterior-posterior axis forms • the dorsal-ventral axis is not determined until fertilization • once the A-P and D-V axes are established – the bilateral left-right axis is fixed

• upon fusion of the egg and sperm - the egg surface (plasma membrane and inner cortex) rotates with respect to the inner cytoplasm – called cortical rotation – always toward the point of sperm entry • rotation brings molecules from the cytoplasm of the animal hemisphere to interact with molecules in the cortex of the vegetal pole – inductive interactions activate regulatory factors in the vegetal cortex – changes gene expression in that region – leads to expression of dorsal- and ventral-specific gene expression Dorsal Right Anterior Posterior Left Ventral (a) The three axes of the fully developed embryo Animal hemisphere Animal pole Vegetal hemisphere Vegetal pole (b) Establishing the axes Point of sperm nucleus entry Gray crescent Pigmented cortex Future dorsal side First cleavage

• in chicks - gravity is involved in establishing the anterior-posterior axis – as the egg travels down the oviduct prior to being laid • later, p. H differences between the two sides of the blastoderm establish the dorsal-ventral axis – if the p. H is reversed above and below the blastoderm – reverses cell’s fates – the side facing the egg white becomes the ventral part of the embryo – the side facing the yolk becomes the dorsal part • in mammals - experiments suggest that orientation of the egg and sperm nuclei before fusion may help establish embryonic axes

Restricting Developmental Potential • Hans Spemann performed experiments to determine a cell’s developmental potential (range of structures to which it can give rise) • embryonic fates are affected by distribution of determinants and the pattern of cleavage • the first two blastomeres of the frog embryo are totipotent (can develop into all the possible cell types) – cells in the inner cell mass of a human embryo are also totipotent Control egg (dorsal view) 1 a. fertilized salamander eggs allowed to divide normally gray crescent in between Gray crescent the first two blastomeres 1 b. fertilized eggs constricted by a thread -shifted the gray crescent toward one blastomere 2. two blastomeres separated and allowed to develop RESULTS: blastomere that didn’t receive any material from the gray crescent – loss of dorsal structures Normal 1 a Control group Experimental egg (side view) 1 b Experimental Gray group crescent Thread 2 Belly piece Normal

Cell Fate Determination and Pattern Formation by Inductive Signals • Question: when can cell fate can be modified? • in mammals - embryonic cells remain totipotent until the 8 -cell stage – much longer than other organisms • BUT - progressive restriction of developmental potential is a general feature of development in all animals • in general: tissue-specific fates of cells are fixed (i. e. determined) by the late gastrula stage • as embryonic cells acquire distinct fates - they influence each other’s fates by induction – conditional specification – i. e. through the production of inductive signals

The “Organizer” of Spemann and Mangold • • Spemann and Mangold transplanted tissues between early gastrulas and found that the transplanted dorsal lip triggered a second gastrulation in the host the dorsal lip functions as an organizer of the embryo body plan, inducing changes in surrounding tissues to form notochord, neural tube, and so on – production of a growth factor called BMP 4 is critical for early embryogenesis • causes the development of ventral structures – involved in cell fate determination and specification – Spemann’s Organizer works to inactive BMP 4 signaling on the dorsal side of the embryo • determines what will be dorsal and what will be ventral EXPERIMENT Dorsal lip of blastopore RESULTS Primary embryo Secondary (induced) embryo Pigmented gastrula (donor embryo) Nonpigmented gastrula (recipient embryo) Primary structures: Neural tube Notochord Secondary structures: Notochord (pigmented cells) Neural tube (mostly nonpigmented cells)

Induction: Formation of the Vertebrate Limb • Inductive signals also play a major role in pattern formation – the development of spatial organization • the molecular cues that control pattern formation are called positional information – this information tells a cell where it is with respect to the body axes – it also determines how the cell and its descendants respond to future molecular signals

Drosophila Embryogenesis early pattern formation work done in Drosophila – translates to higher organisms – three kinds of pattern formation genes • 1. maternal effect genes – genes of the oocyte – – responsible for the axes of the embryo genes translated make proteins that form gradients within the embryo protein gradients are created using the cytoskeleton e. g. concentration of Bicoid protein at anterior end of the embryo results in head structures • 2. segmentation genes – establish the segmented body plan from A to P • 3. homeotic genes – produce proteins with a highly conserved DNA binding region (homeodomain) – found on chromosome 3 in a series of clusters (multiple HOX genes per cluster) – control A-P axis formation along with maternal effect and segmentation genes – after the segments form along the AP axis – the hox genes determine what type of segments they will become » Antennepedia cluster of HOX genes leg formation -HOX genes are very active in human emrbyogenesis

• • • the wings and legs of chicks, like all vertebrate limbs, begin as bumps of tissue called limb buds one limb bud–regulating region is the apical ectodermal ridge (AER) the second region is the zone of polarizing activity (ZPA) Anterior Limb bud AER ZPA Limb buds 2 Posterior 50 m Digits Apical ectodermal ridge (AER) Anterior 3 4 Ventral Proximal Distal Dorsal Posterior (a) Organizer regions (b) Wing of chick embryo

• apical ectodermal ridge (AER) – thickened ectoderm at the bud’s tip – removal blocks the outgrowth of the limb along the proximal-distal axis – AER secretes growth factors in the FGF family • zone of polarizing activity (ZPA) – mesodermal tissue under the ectoderm where the posterior side of the bud is attached to the body – necessary for pattern formation along the anterior-posterior axis – e. g. in humans – determines where thumb and 5 th digit are positioned – cells nearest the ZPA become the most posterior of the digits Anterior Limb bud AER ZPA Limb buds 50 m 2 Posterior Digits Apical ectodermal ridge (AER) Anterior Ventral Proximal Dorsal (a) Organizer regions 3 4 (b) Wing of chick embryo Distal Posterior

• tissue transplantation experiments support the hypothesis that the ZPA produces an inductive signal that conveys positional information indicating “posterior” EXPERIMENT Anterior New ZPA Donor limb bud Host limb bud ZPA • inductive signal of the ZPA was found to be Sonic hedgehog – also involved in segmentation in the fruit fly (called hedgehog) – important roles in humans too – SHH gradient results in the limb - decreasing levels of SHH determines anterior Posterior RESULTS 4 3 2 • BUT before that: Hox genes also play roles during limb pattern formation – Hox gene expression determines whether a limb will be a forelimb or a hindlimb – so HOX and SHH determine arms vs. legs and toes vs. fingers 2 4 3

Cilia and Cell Fate • Ciliary function is essential for proper specification of cell fate in the human embryo • two kinds of cilia – 1. Motile – 2. Non-motile – monocilia • found on nearly all cells • motile cilia play roles in left-right specification – creation of inductive signal gradients within the embryo? redistributes cells within the embryo – study of certain developmental problems called Kartagener’s syndrome • non-motile sperm in males, sinus infections and bronchial infections – motile cilia no longer work • situs inversus - reversal of normal left-right symmetry – right to left flow by cilia creates an asymmetry between left and right halves of the body • monocilia play roles in normal kidney development – function as an “antenna” for multiple signaling molecules