Vertebrate Embryology BioS 325
Lecture Notes

Week 1-2

1. Vertebrate Embryology - development from the fertilized egg to hatching/birth in Vertebrates (fish, amphibians, reptiles, birds and mammals).

2. Development - process whereby cells form tissues, organs (organogenesis) and body structures and acquire specialized characteristics (determination and differentiation).

3. Determination - progressive programming due to the expression of specific subsets of regulatory genes which gradually channels cells to specific pathways of development. Cells are totipotent until gastrulation, then become multipotent and finally unipotent due to the progressive programming.

4. Differentiation - readout of the program leading to a specialized cell with a specific physiological/structural function (ex myocyte, hepatocyte, keratinocyte, RBC).

5. Conservation of developmental processes:
- Developmental Sequences: gametogenesis, fertilization, blastulation, gastrulation Æ ectoderm, mesoderm and endoderm.
- DNA, genes, organelles and biochemical pathways (cell cycle genes).
- Regulatory genes (ex. homeobox genes such as PAX6) code for proteins which bind to other genes and determine whether they will be repressed or expressed.

6. Sexual Reproduction Ð genetic variability via mutation, recombination, and random assortment which produces new combinations of genes. Parthenogenesis (development without mating or fertilization) occurs in few vertebrates.

7. Spermatogenesis - Sperm are formed in the seminiferous tubules of the testis and are required to activate the egg, restore the diploid chromosome number and provide the egg with a centriole. Interstitial cells of Leydig, located between seminiferous tubules, secrete testosterone.

8. Primordial Germ Cells arise in the yolk sac and migrate to the developing gonad to form spermatogonia. Until puberty only spermatogonia and Sertoli cells are located in the seminiferous tubules.

9. Spermatogonia are diploid (XY) stem cells. They will form primary spermatocytes which go through meiosis (duplicate their chromosomes, XY Ð XYXY, and go through 2 meiotic divisions to produce 4 sperm). The 1st meiotic division yields 2 haploid (XX or YY) secondary spermatocytes. The 2nd produces 4 spermatids (X or Y).

10. The seminiferous epithelium is organized into 2 compartments (mitotic/DNA duplication compartment and meiotic division compartment) by tight junctions between Sertoli cells. Most epithelia have a basal lamina and free surface (ex the lining of various organs), are packed with cells (in contrast to connective tissue have very little extracellular material), usually do not have blood vessels or nerves.

11. RH neurons, which originate in the olfactory epithelium, migrate to the hypothalamus. They control the release of gonadotropins (follicle stimulating hormone and lutenizing hormone) from the pituitary gland. FSH acts on the Sertoli cell and LH on the Interstitial cells of Leydig.

Weeks 2-3

1. Just as sperm develop in close contact with Sertoli cells, eggs (oocytes or oocytes with egg coating) are "nursed" through their development by follicle cells. Like Sertoli cells, follicle cells also respond to FSH since they have FSH receptors.

2. Oogonia are converted to Primary Oocytes, arrested in prophase of the 1st meiotic division, before birth. The primary oocyte and the single layer of flat follicle cells which surround it form the Primary Follicle.

3. Follicle cells and the Oocyte are separated by a transparent structure, the Zona Pellucida, and communicate via microvilli which project through the zona.

4. The transition from a Primary Follicle to a Secondary Follicle, beginning at puberty, is mediated by FSH and locally produced Activin which cause the follicle cells to proliferate and follicular fluid to collect in a follicle cavity.

5. Near the middle of the ovarian cycle, just prior to ovulation, the growing follicle becomes a mature Graafian Follicle.

6. The midcycle surge of LH stimulates the primary oocyte to complete the first meiotic division ' secondary oocyte + 1st Polar Body. Shortly thereafter LH stimulates ovulation of the secondary oocyte with the surrounding follicle cells and in the ovary, causes lutenization of the outer wall of the Graafian follicle ' Corpus Luteum.

7. The theca interna (like Leydig cells) have LH receptors and secrete androgens such as testosterone in response to LH. The androgens are taken up by follicle cells and converted to estrogens.

8. Follicle cells release estrogen (E) before ovulation and estrogen and progesterone (P) after ovulation. These steroids regulate the uterine menstrual cycle.

9. E stimulates regrowth of the uterine endometrium and E + P induce the glands of the endometrium to become engorged and secrete nutrients for the developing embryo if fertilization and implantation occurs.

10. If the secondary oocyte is not fertilized, the corpus luteum degenerates and the consequent drop in E+P brings on the menstrual flow.

11. If the secondary oocyte is fertilized and implants the chorion of the embryo secretes a hormone HCG (human chorionic gonadotropin) which maintains the corpus luteum for 4-6 months.

12. The corpus luteum of pregnancy secretes the high levels of E+P necessary to maintain pregnancy and inhibit FSH and LH production by the pituitary (basis of birth control pill). The placenta then takes over the production of E+P.

Week 3

1. Sexual reproduction involves the production of haploid gametes from 2 individuals and their fusion at fertilization to produce an unique 1-celled individual.

2. To successfully accomplish fertilization sperm must be capacitated and find the recently ovulated egg in the upper part of the oviduct. This probably involves signals from the egg or its surrounding coatings which guide the sperm to the egg.

3. Sperm must swim past the loose follicle cells and penetrate the zona pellucida before the sperm and egg cell membranes contact each other and fuse.

4. When sperm contact the zona they begin hyperactive swimming and undergo the acrosome reaction, the release of acrosomal enzymes necessary to digest the zona.

5. Sperm-zona recognition requires the interaction of specific proteins of the zona (ZP-3) with receptors on the sperm head.

6. Sperm-egg recognition also involves a specific interaction between cell membrane proteins of the sperm and egg.

7. Fusion of the egg and sperm cell membranes initiates 2 blocks to polyspermy - a fast block due to depolarization of the egg membrane, caused by a rapid influx of Na+ and efflux of H+ and a slow block due to the release of Ca+ which causes release of cortical granules carrying enzymes which modify the zona pellucida.

Week 4

1. In Vitro Fertilization (IVF) and cloning have challenged our long-standing views of parenthood, family, and personal identity. Individuals conceived in this way may have 3 mothers, virgin mothers, mothers who are also their grandmothers. 3rd party sperm and eggs, embryo adoption and surrogacy are alternatives available to overcome infertility.

2. Ethical and legal issues may arise concerning the disposition of frozen embryos.

3. To obtain eggs, hormones such as human menopausal gonadotropin (HMG), synthetic RH, or drugs (clomiphene) are used to increase FSH concentration in the blood. HCG is injected to initiate the final egg maturation and ovulation will occur about 36 hr later.

4. Fertilization of the bird egg (yolk) occurs in the upper oviduct prior to the addition of the albumin and shell. Due to the yolk only a small disc of cytoplasm at the pole of the egg, the blastodisc, cleaves to produce the first layer of cells, the blastoderm.

5. The blastoderm is converted to a blastula embryo with a blastocoel surrounded by the epiblast and hypoblast.

6. The inside portion of the blastoderm, the area pellucida, develops into the body of the embryo. The outside portion forms the area opaca which develops extraembryonic organs.

7. Gastrulation occurs as cells move over the epiblast forming the primitive streak (Hensen's node at the anterior end) and move inside the embryo forming a solid mass of cells which is resolved into the ectoderm, mesoderm and endoderm as the primitive streak regresses to the posterior region of the area opaca.

8. The mesoderm is subdivided into the head mesoderm, notochord, somitic, intermediate and lateral plate mesoderm. Further subdivision of the somites ' dermatome, myotome and sclerotome and subdivision of the lateral plate mesoderm ' somatic and splanchnic mesoderm layers surrounding the coelom.

9. As the primitive streak regresses from anterior to posterior, neurulation of the ectoderm follows producing the primitive neuroepithelial cells of the future CNS and PNS.

10. Due to their size and accessibility, amphibian eggs are an excellent model for studying early development, especially dorsal-ventral axis formation and mesoderm induction.

11. The entire egg cleaves (holoblastic-vs- meroblastic) although the cleavages are slower in the yolky vegetal hemisphere of the egg. Cleavage is rapid producing thousands of cells in a few hours.

12. At fertilization the cortical cytoplasm of the egg rotates about 30 degrees relative to the internal cytoplasm. This shift activates dorsal determinants (maternal factors) which will later specify the dorsal-ventral axis of the embryo.

13. The dorsal region forms opposite the point of sperm entry as the activated determinants induce the marginal cells of the blastula to become committed to mesoderm and migrate into the embryo during gastrulation.

14. The dorsal lip of the blastopore (Spemann organizer), which is homologous to Hensenís node, serves as an organizing center. DL cells will produce the head mesoderm and notochord.

15. Proteins such as "noggin", secreted from the dorsal lip, are involved in the induction of mesoderm such as head mesoderm which induces a head to form.

Week 5

1. Gestation lasts 38 weeks (3, 3 and 3.5 month trimesters). For the first 8 weeks the conceptus is called an embryo and the last 30 weeks a fetus. Most tissues and organs are formed during the embryo stage but do not mature and become functional until the fetal stage.

2. Week 1: After fertilization, the human 1-celled embryo begins to cleave as it is carried down the oviduct toward the uterus. Cleavage of all mammalian fertilized eggs is very slow compared to other vertebrates.

3. Compaction results from the formation of tight junctions between the outer blastomeres. The outer blastomeres pump fluid into the embryo forming a fluid-filled blastocoel cavity. When the blastula embryo, called a blastocyst, reaches the uterus, it hatches from the zona pellucida and begins implantation into the wall of the endometrium.

4. The blastocyst is composed of an inner cell mass, which will form the embryo and some extraembryonic organs (amnion, yolk sac and allantois) and the trophoblast which forms the chorion of the placenta.

5. Implantation is mediated by the syncytiotrophoblast, a syncytium derived from the cellular trophoblast (cytotrophoblast).

6. Week 2: The inner cell mass is converted to a bilayered embryo - epiblast and hypoblast. The hypoblast forms the endodermal lining of the yolk sac. The epiblast forms the ectodermal lining of the amnion. Extraembryonic mesoderm (origin controversial) forms the mesoderm of the amnion and yolk sac.

7. Week 3: Gastrulation occurs in the epiblast producing a tri-layered embryonic disc. Ectoderm, endoderm and mesoderm are formed as cells move through the primitive streak as it regresses towards the posterior region of the epiblast. Extraembryonic mesoderm from the primitive streak provides support and a site for blood vessels to provide nutrients for the epithelial lining of all the extraembryonic organs (amnion, chorion, allantois, yolk sac).

8. The notochord, characteristic of all Vertebrates (and other chordates), provides support and induces the neural plate to form in the overlying ectoderm. It also induces the ventral neural tube to become floor plate and somitic cells to become sclerotome.

9. Week 4 &endash; 8: Neurulation produces the CNS from the neural tube and PNS from the neural crest. Major subdivisions of the mesoderm form and the flat disc-shaped embryo folds to form a 3D tubular embryo with separate intraembryonic and extraembryonic regions. Numerous organs form (organogenesis) including the heart and the organs of the urogenital system and gastrointestinal system.

10. By the end of the 8th week the embryo is about 1 inch in length and major changes in it's external features such as the formation of the limbs, ears, eyes and face allow it to be recognized as human &endash; thereafter it is called a fetus.

Week 5-6

1. The ectoderm produced at gastrulation has the potential to form the nervous system as well as the epidermis.

2. Transplantation studies have demonstrated that the decision of ectoderm to enter the nervous system pathway, rather than the epidermis pathway, occurs as the head mesoderm and notochord form during gastrulation (see handout).

3. Presumptive epidermis can be induced to form brain by anterior mesoderm and removal of pieces of mesoderm results in the failure of the overlying section of nervous system to form, indicating that formation of the nervous system is dependent on inductive signals from the underlying mesoderm.

4. The mesoderm is initially programmed by signals (possibly activin) emanating from vegetal cells at the late blastula stage. These signals induce the overlying marginal cells at the equator of the blastula to express specific genes and move to form the blastopore (Fig 4-9).

5. Two of the proteins secreted from the dorsal lip (Hensen's node or primitive node), and then in the notochord cells derived from the dorsal lip, include noggin and chordin.

6. Numerous experiments indicate that these proteins are important inducers of the nervous system in the overlying ectoderm and help explain the ability of a transplanted dorsal lip to induce an entire new anterior-posterior axis in frogs, birds and mice (Fig 4-8).

7. The neural inducers cause the overlying ectoderm to form the neural plate and fold into the neural tube. The cells at the crests of the neural folds form the neural crest cells (Fig 5-11).

8. When the neural plate first appears, its anterior region is already programmed to form the brain and its posterior region will become the spinal cord. This A-P development of the nervous system is due to the A-P positional identity of the underlying head mesoderm and somites, which in turn is specified by homeobox gene expression.

9. Homeobox genes (ex Lim-1, Fig 4-11) code for regulatory proteins, that control gene expression by binding to other genes and controlling whether they are expressed or repressed.

10. Signals from the notochord are also important for inducing the floor plate of the neural tube (which is required for the formation of motor nerves) and the sclerotome of the somites, which form the vertebrae surrounding the spinal cord (Handout).

11. Somites are formed from somitomeres. The anterior 7 pairs of somites do not form, they remain somitomeres (Fig 5-16).

12. The anterior and posterior halves of the sclerotomes of adjacent somites form the body of the vertebrae and spinal nerves grow out between the vertebrae where the muscles extend over the intervertebral joints (Figs 5-17, 5-19).

Week 7

1. Extraembryonic organs (membranes) are required for embryos to survive and grow prior to birth or hatching.

2. The amnion is an adaptation to terrestrial life found in reptiles, birds and mammals. It prevents the embryo from drying out, serves as a shock absorber and prevents adhesion between the growing embryo and its surrounding structures.

3. The chorion is the outermost layer which protects the embryo and interacts with the environment. In combination with the allantois it forms the placenta and functions in respiration (chorio-allantoic membrane).

4. The allantois stores waste products in reptiles and bird eggs. Its mesodermal component forms the umbilical arteries and veins and the blood vessels of the chorio-allantoic membrane.

5. The yolk sac is the site of origin of primordial germ cells and stem cells of the blood. In birds and reptiles it provides nutrients for the growth of the embryo.

6. The amnion and chorion are formed by the somatopleure during the folding which converts the embryo from a flat disc shape to a 3D tubular form. The allantois and yolk sac are derived from the splanchnopleure.

7. The head fold forms the foregut and the tailfold forms the hindgut. The midgut decreases in length as the fore- and hind-gut increase in length. Eventually the foregut forms the pharynx, esophagus and stomach; the midgut forms the small intestines and a part of the colon; and the hindgut forms the remainder of the colon and cloaca.

8. The oropharyngeal and cloacal membranes separate the endodermal lined gut from the ectodermal lined stomodeum and proctodeum.

9. Inpockets of the ectoderm (pharyngeal grooves) and outpockets of the endoderm (pouches) divide the lateral regions of the pharnyx into 4-5 pharyngeal arches. Each arch is innervated by a cranial nerve, supplied by an aortic arch and forms specific skeletal elements and muscles.

10. The mesenchyme cells of the arch have a dual origin - from the head somitomeres and neural crest.

11. The 1st pharyngeal arch is innervated by the trigeminal nerve and forms the upper and lower jaws and chewing muscles. The 1st aortic arch is formed as the head fold bends the heart and dorsal aortae ventrally. The 1st pharyngeal groove forms the external auditory meatus and the pouch forms the eustachian (auditory) tube.

12. The tonsils, thymus and parathyroids develop from the other pharyngeal pouches and the thyroid develops from the floor of the pharynx.

Week 8

1. The first heart is a simple tubular structure composed of 4 regions (sinus venosus, atrium, ventricle and bulbus cordis). This simple tube, which carries both arterial (oxygenated) and venous (deoxygenated) blood, must be converted to the adult heart with 4 chambers and separate venous and arterial blood pathways in parallel.

2. The simple tubular heart develops from splanchnic mesoderm cells which initially form in the anterior, dorsal region of the embryonic disc.

3. These cells form 2 endocardial tubes which are moved ventrally by the head fold and fuse to form the endocardium (inner lining of heart), after the foregut is formed. The splanchnic mesoderm becomes the myocardium (muscle) which produces an acellular, spongelike mass called the cardiac jelly. The epicardium (outer connective tissue) is formed by migration of caudal splanchnic mesoderm cells.

4. The conversion of this simple tubular heart to a 4 chambered heart is accomplished by looping and partitioning of the atrium, ventricles and bulbus cordis.

5. The atrium is partitioned by 2 septa (septum primum and secundum), each of which have a foramen that allows blood to flow between the right and left atria.

6. The outflow tract (bulbus cordis) is derived from paraxial and lateral mesoderm and neural crest (not splanchnic mesoderm). As bulbus cordis elongates, 2 regions can be recognized, the conus arteriosus and truncus arteriosus. It is then partitioned by spiral trunco-coronal ridges into the aortic and pulmonary blood vessels which drain the left and right ventricles.

7. The sinus venosus is incorporated into the wall of the right atrium and in part becomes the sino-atrial node. The SA node and the later forming atrio-ventricular (AV) node, the heart pacemakers, regulate heart beat by sending a contractile stimulus to the atria and ventricles via conducting fibers of modified cardiac myocytes.

8. At birth, the interatrial foramen and the shunts (ductus arteriosus and ductus venosus) which allow blood to bypass the lungs and liver are closed off.

9. The kidney is composed of functional units called nephrons (tubules), glomeruli, tightly wound balls of blood capillaries which bring metabolic wastes to the nephrons and excretory ducts which carry urine to the bladder. The nephrons and ducts develop from intermediate mesoderm while glomeruli develop from dorsal or lateral mesoderm.

10. The kidney functions by removing nitrogenous wastes and controlling blood volume, pressure and acid/base (pH) balance. It does this by concentration, selective absorption and elimination of nitrogenous wastes, water and various ions.

11. The 3 types of kidneys found in vertebrates are the pronephros, mesonephros and metanephros. They develop from anterior, middle and posterior regions of intermediate mesoderm.

12. Kidney development (ontogeny) in birds, reptiles and mammals recapitulates, in part, the evolutionary history (phylogeny) of the kidney in vertebrates - the 3 types of kidneys develop in the same ordered sequence as they appeared during evolution.

13. The fluid portion of blood is filtered from the glomerulus and enters the nephron (Bowman's capsule). After the glomerular filtrate is processed the concentrated urinary wastes pass into the excretory duct system of the kidney. Then urine is transported to the urinary bladder via the ureters and eliminated via the urethra.

14. The mesonephric duct develops from the anterior nephrotomes and grows toward the cloaca. As the duct passes the middle portion of intermediate mesoderm, it induces the formation of 1 or a few nephrons per segment which become the mesonephric kidney.

15. The metanephric kidney is induced by the ureteric bud, an outgrowth of the caudal part of the mesonephric duct. As the ureteric bud grows into the metanephrogenic blastema, it goes through a series of branchings to form the ureter, the renal pelvis, the major and minor calyces and millions of collecting ducts. The collecting ducts then induce millions of nephrons making the metanephric kidney a very efficient organ compared to the mesonephric kidney.

16. The cloaca is partitioned by the urorectal fold into a dorsal rectum and ventral urogenital sinus.

17. The mesonephric duct and ureter are adsorbed to the portions of the urogenital sinus which become the urethra and bladder.

Week 9

1. The complexity of the nervous system is staggering. The body is festooned with a network of nerves whose signals are processed and coordinated through the central nervous system, allowing us to respond to sensory signals, store and recall information and control numerous body functions. Cranial and spinal nerves originate in brain and spinal cord (CNS) as well as the spinal ganglia and autonomic ganglia of the peripheral nervous system (the PNS).

2. The CNS develops from the neural tube. All spinal ganglia and most cranial ganglia develop from neural crest. Some cranial ganglia are derived from ectodermal placodes.

3. Cranial and spinal nerves are bundles of fibers (axons and dendrites) which carry signals to the CNS (sensory fibers) and/or away from the CNS (motor fibers).

4. When the neural tube first forms it is composed of a single cell type, the neuroepithelial cell. These cells are arranged in a simple columnar epithelium with external and internal limiting membranes. Their nuclei move from one end of the cell to the other as they go through the cell cycle, so the epithelium appears to be stratified.

5. As cells stop dividing and begin to differentiate they lose their connection with the internal limiting membrane. The neuroblasts are the first cells to differentiate and occupy the intermediate zone of the neural tube. This zone contains the cell bodies of neurons and is shaped into dorsal and ventral horns. Neurons in these horns (columns) develop sensory and motor fibers, respectively.

6. The neuroblasts send out axons and dendrites toward the external limiting membrane. This outer zone is called the marginal zone. Glial cells differentiate next and act as nurse cells for the neurons. They support nerve fibers and wrap their cell membrane around them forming the myelin sheaths. The innermost layer of cells constitutes the ventricular zone. They develop into ependymal cells which line the fluid filled central canal of the spinal cord and ventricles of the brain.

7. The 30 spinal nerves are all mixed, they carry motor and sensory fibers of the voluntary, somatic NS and motor fibers of the involuntary, autonomic NS (sympathetic and parasympathetic).

8. From touch or pain receptors in the skin nerve, impulses are carried via sensory fibers to the dorsal horn of the spinal cord. The signal is then sent by interneurons which synapse with motor neurons in the ventral horn. The motor neurons carry the signal to nearby muscles completing the simple reflex arc.

9. The motor fibers of the somatic NS grow out of neuronal cell bodies in the ventral horn of the spinal cord. The motor fibers are combined with sensory fibers growing out of the cell bodies of the spinal ganglia to form a spinal nerve.

10. At levels from T1 to L2, preganglionic motor fibers of the sympathetic autonomic system join the spinal nerves. These preganglionic motor fibers originate in the intermediate horn of the spinal cord and synapse with postganglionic motor neurons in the sympathetic ganglia. They control involuntary reactions to stress.

11. Pre- and post-ganglionic motor fibers of the parasympathetic autonomic system join some spinal nerves, but only in the sacral region of the spinal cord. Preganglionic fibers of the parasympathetic system also originate in the brain and are carried in cranial nerves to ganglia located in various organs. They synapse with postganglionic fibers and control involuntary reactions in non-stress, resting situations.

Week 10-11

1. What makes us male or female? - sex chromosomes, testosterone and estrogen, external genitalia, secondary sex characteristics, ability to be pregnant and give birth, communication skills, spatial and verbal skills, aggressive tendencies?

2. However, certain individuals (male pseudohermaphrodites) with a Y chromosome, testes and testosterone are female. These individuals develop as females because of a mutation in the receptor for testosterone prevents male sex differentiation.

3. Sex determination and sex differentiation are controlled by regulatory genes and hormones.

4. Sex determination begins with the expression of regulatory genes in the indifferent gonad. The genes expressed determine if the indifferent gonad will develop into either a testis or ovary.

5. A testis will normally form if a Y chromosome gene (Sry/TDF=testis determining factor) is expressed in the cells of the sex cords.

6. The genetic program leading to an ovary will be activated automatically unless Sry/TDF is expressed.

7. Sex differentiation is induced by testosterone and estrogen which cause the differentiation of male/female ducts, glands, external genitalia, body shape, secondary sex characteristics and behavior.

8. Intersexes have some male and some female reproductive organs.

9. The most important reproductive organ, the brain, controls gender identity, sex preferences and sex specific behaviors.

10. A continum between male and female exists in reproductive anatomy and most likely in brain gender identity.The case of John/Joan indicates that gender ID is controlled by both genes and environment.

11. The TDF gene was located in humans by correlating maleness or femaleness with the presence or absence of a specific region of the Y-chromosome in XX males and XY females. Subsequent sequencing of this region identified the TDF gene.

12. The various organs of the reproductive system (gonad, ducts, glands, external genitalia) are bipotential, they can enter the male or female pathway of development.

Week 12

1. The skin is composed of an epidermis, a stratified epithelium derived from ectoderm, and the underlying dermis, connective tissue derived from the dermatome, somatic mesoderm and neural crest.

2. Proliferation of the ectoderm leads to the gradual stratification of the epidermis. When the cells lose their attachment to the basal lamina they differentiate as they move toward the outer surface. The main luxury protein are keratins and the differentiating cells are called keratinocytes.

3. The basal layer of cells include unipotent stem cells and progenitor cells. Stem cells divide throughout life but very infrequently. Progenitor cells divide frequently but unlike stem cells, are destined to differentiate after a finite number of divisions.

4. Hematopoietic stem cells of the bone marrow are mutipotent since they are capable of producing all types of blood cells. They divide infrequently and give rise to rapidly dividing progenitor cells. Progenitor cells become restricted in developmental potential and then differentiate into one specific type of blood cell before entering the peripheral circulation.

5. Stem cells of adult organs show unexpected flexibility when transplanted into new environments. They differentiate into cell types that they would not normally form, i.e. rare (1 in 10 billion) bone marrow cells have been demonstrated to give rise to neurons, muscle and liver cells.

6. Mouse embryo stem cells are derived by culturing the inner mass cells of the blastocyst on feeder layers of fibroblasts. They are mutipotent and divide continuously without differentiation when specific growth factors are added to the growth medium.

7. ESC will differentiate in response to specific growth factors, i.e. retinoic acid routes them into glial or neuronal paths of differentiation. They have been used for 20 years to study determination and differentiation in vitro.

8. Human ESC lines have been developed recently from blastocyst inner mass cells and fetal primordial germ cells. Work has now begun to get them to differentiate in vitro so they can be used for transplant experiments to replace dying cells and regenerate functional tissues and organs.

9. Mouse ESC lines are currently being used to develop these transplantation procedures. Experiments with mouse ESC have been shown that they partially restore spinal cord function in rats paralyzed by spinal injuries and are capable of myelinating nerve fibers in rats with demyelinating diseases.

10. Besides all the issues associated with transplanting the correct number of cells in the exact location and getting them to function properly, ESC form tetatomas and are subject to elimination by the hosts immune system.

11. Fetal brain cells have been used over the past 10 years to treat individuals with Parkinson's disease. Replacing dying dopamine&endash;producing cells of the brain with fetal cells has led to an improvement in control of movement of about 50% in some cases. Grafting the right number of fetal cells in the right places of the brain is difficult and will be improved if ESC can be converted to dopamine&endash;producing cells.

12. The ethics of isolating ESC from human blastocyst embryos are currently being discussed. The National Bioethics Advisory Commission has recommended that researchers funded by the government should not create new cell lines, but can carry on research with ESC lines produced by private firms.

13. Adult stem cells and ESC are also being used to form organs. The stem cells are seeded in artificial biodegradable matrices in the shape of the organ to be duplicated. Skin and bladders have been produced.

Week 13

1. Clones are genetically identical. Molecular biologist clone genes and cell clones are produced by cell division. Virtually all the cells of our body are clones since they have the same genes. Genetically identical individuals are also clones. They only have the genes of one individual and therefore cloning is a form of asexual reproduction.

2. Genetically identical plants and animals are produced in various ways in nature. Identical human twins are clones.

3. When people speak of cloning however they are not generally thinking about naturally produced clones, they are thinking of clones produced by nuclear transplantation - where a nucleus of a somatic cell is introduced into an enucleated egg.

4. Cloning of animals may be done to make multiple copies of valuable animals. In that case the somatic cell nucleus comes from a valuable adult.

5. A less controversial form of cloning is embryo splitting. A newborn rhesus monkey was recently produced from a quarter of an embryo (obtained by separating an 8-cell embryo into four 2-cell embryos). Since the cells cloned are from an embryo, we have no idea if they are valuable or what their phenotype will be.

6. The first animals to be cloned were frogs. Biologist cloned frogs to study determination and differentiation.

7. It was discovered that cloning was more difficult as the developmental age and state of differentiation of the embryo increased. It is relatively easy to clone a blastula embryo by transferring a nucleus from one of its cells to an enucleated egg - 30% developed to adults. When cloning was attempted from cells of gastrula, neurula, tadpoles or adults, fewer and fewer successful clones could be obtained. In fact a cell from an adult frog has never been cloned!

8. These results demonstrated that genes are not lost or permanently altered during differentiation. Reprogramming the genes of a somatic cell nucleus occurs in the cytoplasm of the egg. Many maternal factors are know to be present in the egg cytoplasm and these factors enter the somatic cell nucleus, just as they would enter the zygote nucleus, and control gene expression required for early development.

9. Mammals have been cloned using a more gentle method of transferring the somatic cell nucleus to the enucleated egg. By fusing the cell membranes of the somatic cell and egg, chromosome damage is avoided and it is possible to clone cells of adult animals. Dolly was the first animal cloned from a adult cell, in her case a mammary gland cell.

10. Since mammalian development needs a uterine environment, cloning mammals requires the procedures developed for in vitro fertilization. After the single cell embryo is grown to the morula or blastocyst stage it is transferred to the uterus.

11. Subsequent work has show that mammalian clones have mitochondria derived from the somatic cell as well as from the egg. Telomere length has been reported to reflect the age of the donor cell but Dolly does not appear to have prematurely aged.

12. The somatic cells which will be cloned can be genetically modified by introducing genes of any species. These cells are then used to create transgenic animals.

13. Mammals will be cloned to reproduce valuable animals, i.e. cows which produce large quantities of milk, goats or sheep that carry human genes coding for biomedically important proteins, animals which have been genetically modified so that they serve as models for specific human diseases, or as sources for xenotransplants.

14. Currently the best success in cloning has been obtained with mice, about 2% of the embryos created developed to term. The techniques for cloning humans are readily available but we do not know how successful the process will be. The high number of embryo and fetal deaths and individuals born with developmental defects indicates that it would be medically unsound and unethical to try to clone humans at this time.

15. There are good reasons for cloning animals but the question still remains, "Why should we clone humans?" Because we can? To make multiple copies of great athletes or intellects? Provide humans for organ transplants and research? Replace children or spouses who have died? Help infertile couples?

Week 14

1. Cancer cells are derived from normal cells by mutation, the action of tumor promoters, which do not cause mutations, and in some cases by viruses.

2. The conversion from normal cells to cancer cells includes changes in 4 cell behaviors.
1) Controlled cell proliferation -vs- uncontrolled cell growth
2) Differentiation allows function -vs- No differentiation leads to loss of function
3) Do not cross tissue compartment -vs- metastasis leads to malignancy
4) Do not attract blood vessels -vs- attract blood vessels

3. Loss of growth control and the ability to differentiate produces a benign cancer. The benign cancer will not grow unless it is able to attract blood vessels for nourishment.

4. Metastasis: cells cross tissue boundaries, enter blood or lymph vessels, and then colonize other tissues producing a malignant cancer.

5. A balance between cell birth and cell death achieves the control of cell proliferation in adult tissues. Stem cells produce progenitor cells that differentiate into mature functional cells. These cells perform their function for a given time and then undergo programmed cell death (apoptosis).

6. The control of cell proliferation occurs through the balanced expression of proto-oncogenes, which speed cell division and tumor suppressor genes, which suppress cell division.

7. Mutation of proto-oncogenes and tumor suppressor genes of normal cells produces cancer cells. Mutation of proto-oncogenes (to oncogenes) leads to an over stimulation of cell division; mutation of tumor suppressors prevents them from inhibiting cell division. These mutations lead to the loss of control of cell proliferation.

8. Malignant cancer is the end result of numerous changes in cell behavior (growth, differentiation, migration, obtaining nutrients) brought about by multiple mutations and the selection of cells best adapted to grow, metastasize and continue to evolve. Consequently, cancers take many years to develop.

9. Cancers usually arise in tissues that have the capacity for cell division (ex epithelial cells with dividing populations of stem/progenitor cells).

10. Tumor progression involves mutations as well as cancer promoters that act by altering gene expression, cell multiplication and the selection process.

11. The mutations must not only occur in the correct gene but in a specific part of that gene that alters its function and in the correct tissue, where that gene is being expressed.

12. Mutations that alter DNA replication and repair, cell cycle controls and chromosome dynamics lead to genetic instability - the rapid accumulation of mutations and chromosome damage that increases the rate of tumor progression.

13. Like hair follicles and glands derived from the epidermis, mammary glands develop by a inward growth and branching of ectoderm, controlled by cell signaling from the underlying connective tissue.

14. Breast cancers arise from the epithelial cells of milk ducts and glands. They are classified as sporadic (90%), which are due to random mutations in individuals without a family history of breast cancer, or inherited (10%), due to the inheritance of mutant genes such as BRAC1 or BRAC2.

15. BRAC 1 & 2 are regulatory genes (transcription factors) that are involved in the control of DNA replication and repair, cell cycle progression and apoptosis. When both copies of BRAC1 are inactivated by mutation they lose their normal function as tumor suppressors leading to mutations of genes such as p53 and genetic instability.

16. Estrogen acts as a promoter of breast cancers by stimulating proliferation of cells expressing the estrogen receptor. Tamoxifen and Raloxifene inhibit the estrogen effects by binding the estrogen receptor. This non-productive interaction prevents estrogen from binding and inducing cell division.

17. Estrogen acts on many organs including the mammary glands, heart, bones and brain. The estrogen-receptor interactions in these different tissues are slightly different, allowing the development of drugs that block the receptors of breast cells but not those in the heart, etc.

18. Herceptin is an antibody to EGFR (epidermal growth factor receptor) that prevents EGF(epidermal growth factor) from binding and inducing cell multiplication. It is used for cancers that over express EGFR.

19. Over 50% of cancers are due to environmental agents and therefore can be eliminated by changes in life style (smoking, diet, UV exposure). Early diagnosis is critical to prevent metastasis (PAP test, prostate exams, PSA, breast exams, mammography).

Week 15

1. The chorion, amnion, allantois and yolk sac are extraembryonic organs that are required for the in utero growth and development of the embryo.

2. The amnion, which serves to protect the embryo and allow growth and movements, expands to completely surround the fetus. Eventually it eliminates the chorionic cavity (extraembryonic coelom) and forms the outside wall of the umbilical cord.

3. Amnionic fluid is derived from numerous sources but later in pregnancy the fetal kidneys contribute a large portion of it.

4. Prenatal diagnosis can be done by removing amnionic fluid (amniocentesis) sometime after the 13-15 week of pregnancy.

5. The chorion forms chorionic villi, the fetal portion of the placenta. It also serves as an immunological barrier that prevents the mother's immune system from rejecting the fetus.

6. The mesoderm of the allantois also contributes to the fetal portion of the placenta (chorio-allantoic placenta).

7. The placenta is composed of fetal and maternal parts.

8. The fetal portion is made up of branching outgrowths of the chorion called chorionic villi.

9. Initially, chorionic villi arise as outgrowths of the syncytiotrophoblast that degrade endometrial tissue in its path leading to the formation of spaces (trophoblastic lacunae) that fill up with maternal blood.

10. Mature chorionic villi are formed by finger-like outgrowths of cytotrohoblast into the syncytiotrophoblast. A core of extraembryonic mesoderm then grows into these projections and the villi branch extensively into the maternal pool of blood.

11. The mesodermal core of the villus gives rise to the fetal vessels, which connect to the umbilical arteries and vein. The umbilical arteries and veins, which are derived from mesoderm of the allantois, carry fetal blood to and from the fetus via the umbilical cord.

12. Exchange of numerous components including nutrients, gases and waste products (but not blood cells) occurs between the fetal blood in the chorionic villi and the surrounding maternal blood.

13. The maternal portion of the placenta arises from the endometrium and is called the decidua since it is lost as part of the afterbirth (extraembryonic tissues and part of the endometrium).

14. The connective tissue (stromal) cells of the endometrium undergo the "decidual reaction" in response to the invading trophoblast. They accumulate glycogen and lipids that are important for the early growth of the implanting embryo.

15. As the placenta matures the barrier between the maternal and fetal blood thins making exchange more efficient. The syncytiotrophoblast flattens and much of the cytotrophoblast is lost.

16. Besides gasses, nutrient and wastes, bacteria (syphilis), viruses (rubella, cytomegalovirus), drugs, hormones and antibodies can pass through the placenta.

17. Blood cells of the mother and fetus can enter each other's circulation during the birth process. In cases where the mother is Rh negative and fetus Rh positive this can lead to the mother producing Rh positive antibodies which may attack and destroy fetal blood cells.