Developmental Biology Study Guide

Developmental Biology Final Exam Study Guide 12/3/11 Part I Chapter 1 -Basic Problems of developmental Biology: • Maintenance of complete genome while cells differentiate -Main mechanisms of differential gene expression-polarity and cytoplasmic differences; polarity: • Gene expression leads to a difference in cells • Every cell in body has the same genome • Gene regulation occurs: o Polarity and cell division ? Uneven egg contents ? Environmental factors o Gene cascades o Induction ? Cell-cell signaling -What does development encompass? • Development begins at fertilization Study of the emergence of living order -What are the basic questions in developmental biology? 1. Question of differentiation-how can identical genetic instructions produce different types of cells? How can single cell generate so many cells? 2. Question of morphogenesis-how can cells in body organize into functional structures? 3. Question of growth-how do cells know to stop dividing? 4.

Question of reproduction-how are germ cells set apart from somatic cells? What instructions are in the nucleus and cytoplasm? 5. Question of regeneration-how do stem cells retain regeneration capacity?

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Can we harness it to cure diseases? 6. Question of evolution-how do changes in development create new body forms? 7. Question of environmental integration-can environmental chemicals disrupt development? How is development of organism integrated into its habitat? -Some basics: similarity and relatedness of organisms; development of 3 germ layers: • All develop three germ layers: o Ectoderm- skin, nervous system, brain and nervous system o Endoderm- gut, digestive tract and associated organs, the lungs o Mesoderm- all the rest, kidney blood, heart, gonads, bones, muscles History of Embryology-Von Baer’s principles and Haeckel’s misinterpretation; fate mapping and cell lineages: • Von Baer’s Principles: 1. General features appear earlier in development than specialized features 2. Less general characters develop from the more general; until finally the most specialized appear.

(originally have same skin, later skin develops) 3. The embryo of a given species instead of passing through the adult stages of lower animals, departs more and more from them. 4. Therefore, the early embryo of a higher animal is never like a lower animal, but only like its early embryo. Haekel misinterpretated: thought he was talking about recapitulation. (The idea that embryonic development repeats that of one’s ancestors.

) o He implied that we go through a fishlike stage ? We don’t ? However, development (ontogeny) and evolutionary relationships (phylogeny) are related. • Fate maps and cell lineages: o Vital dye marking o Transplantation of labeled cells o Injection of tracers into cells o Chimeras (such as chick-quail) • Some cells are highly migratory -What are the basic cellular processes (ingression, epiboly, etc)? Ingression-migration of individual cells form surface into the interior of the embryo. • Invagination-infolding of a region of cells much like the indenting of a rubber ball when poked. • Involution-inturning or inward movement of an expanding outer layer so that it spreads over the internal surface of the remaining external cells. • Delamination-the splitting of one cellular sheet into two more less parallel sheets.

• Epiboly-the movement of epithelial sheets that spread as a unit to enclose the deeper layers of the embryo. -Homology vs Analogy: Homology=similarity via common ancestry (wing of a bird, arm of a human) • Analogy=via common function (wing of bird and wing of butterfly) -Basic stages of development: 1. Fertilization-fusion of sex cells 2. Cleavage-mitotic divisions 3. Gastrulation-blastomre moves and causes changes in position and results in germ layers 4. Organogenesis-cells interact and rearrange to form tissues and organs 5.

Metamorphisis-changing from one form to another 6. Gametogenesis-development of gametes -Polarity in the egg-animal-vegetal: • Animal pole-“animated” cytoplasm, not yolk Vegetal pole-“vegetated” more yolk, less cytoplasm -Main axes of body symmetry: • Dorsal-Ventral-(back-belly) • Anterior –Posterior-(head-toe) • Left-Right-(side-side) -Classification of metazoan animals: • Multicellular, mitochondrial eukaryotes • Have membrane surrounding nuclear material Chapter 2 -Genetics vs Embryology: • Does the cytoplasm or the nucleus control heredity? o Thomas Hunt Morgan showed nuclear chromosomes responsible for development of inherited traits. • Evidence for nucleus o Polyspermic so embryos abnormal o Role of chromosomes in insect sex determination Embryology argument: o How do the same chromosomes make different cytoplasms? o Do genes really control early development? -Histones; Drosophila polytene chromosomes-heterochromatin vs euchromatin: • Histone-positively charged proteins that are the major component of chromatin. o Maintain the repression of gene expression • Histone acetylation- loosens the histones. ACTIVATES transcription.

• Histone methylation-condenses nucleosomes. REPRESSES transcription. • Polytene Chromosomes-(fly larva) cells replicate chromosomes, but do not divide. Chromosomes become huge and visible. • Heterochromati-inactive genes • Euchromatin-active genes -Cloning and nuclear transplantation: • Nuclear transfer-activation and enucleation of oocyte.

o Transplantation of blastula cell nuclei could direct the development of complete tadpoles when transferred into the cytoplasm of an activated enucleated egg. • Nuclei from adult frogs were not able to generate adult frogs. o Nuclei from adult frogs were able to direct development of all organs of tadpole. • Mammalian cloning: o Cultured adult mammary gland cells. (Held in G1) Enucleated oocytes from a different strain.

o Got Dolly the sheep. • Development directed by the donor nucleus, not the host oocyte. o Cloned animals genetically identical to nuclear donor, but differ in physique and personality. -Differential gene expression: 1. Every cell nucleus contains the complete genome (DNA identical). 2.

The unused genes are not destroyed or mutuated. 3. A small percent of the genome is expressed in each cell (some specific to that cell type). • The control of gene expression is what allows cells to differentiate and form different tissues and organs. Techniques in developmental research: • PCR-heater/cooler o Can isolate a gene o Determine whether gene is expressed in certain time or place.

• RT-PCR-reverse transcription PCR o To get at mRNA expression o Amplification of specific sequence based on complementary primers • In situ-in the embryo o Localization of mRNA w/in the intact embryo o Binding in fixed embryos o Antisense probes complimentary to an mRNA of interest tagged with radioactive or colorimetric marker. o Cells expressing that mRNA will light up. • Gene chip microarray-EXPENSIVE o Allows monitoring of many genes Transcriptional activation at once o Each DNA clone spotted on a side grid • Transgenics-overexpression to get stability o Microinjection o Transfection o Electroporation o Chimeras o Gene knockouts • Antisense- o Function can be impaired by expression o Would bind and inactivate mRNA • Techniques based on centra dogma: o DNA(Transcription(initial RNA transcription(add RNA cap and tail(RNA splicing(completed mRNA(to cytoplasm for translation(protein(stability(modification PART II Chapter 3 -Experimental embryology?????? -Modes of commitment: autonomous vs conditional Mode of specification |Type of Development |Representative organism | |Autonomous |Mosaic |Snail | |Conditional |Regulative |Vertebrae | |Syncytial |Morphogen Gradient |Insect | Autonomous-each cell has an independent fate based on cellular determinatnts. o Cells cannot change fate if blastomroes are lost • Conditional-each cell can give rise to more types than it normally does o Cells can gate o Fate determined late o Depends on cell-cell interaction • Syncytial-cell fate determined by gradients in egg prior to cellularization o Anterior-posterior axis determined -Morphogen gradients and diffusion of morphogens: • Morphogen-a diffusible biochemical molecule that can determine the fate of a cell by its concentration. • Morphogen gradients- Polarity and gradients suggested in regeneration experiments ? Planaria head/tail o Diffusion of soluble substances down a concentration gradient. ? “French Flag Model” • Gradient of morphogen • Gradient of receptor Cascade of Inductions Induction and migration -Cell adhesion: • Cell proteins vary widely • Selective affinity for other cells o One surface of a tissue (+) affinity for one tissue, (-) affinity for the another.

o Affinities may change throughout development. • If cells are clinging together in sheet but cell with a higher affinity comes along, they may “jump ship” and create new sheet. Molecules of cell adhesion: o Cadherins ? Calcium dependent adhesion molecules • Very diverse family of proteins o Transmembrane; anchored intracellularly by catenins ? Functions • Adhesion • Shape change • Gene expression -Cell-Cell communication: • Can signal fate and perhaps indicate the next cells fate • Endocrine-(global) hormones • Juxtacrine-(local) touching • Paracrine-(local) over and area (diffuse) -Embryonic Inductin: • Requires and inducer-signaling to alter the fate of another tissue • Requires competence in the responding tissue The ability to respond to a specific inductive signal. -Competence-ability of cells to receive inductive signals • Gene expression profile in responding cells • May be acquired through earlier signaling events. • What role does competence play in embryonic induction? o Competence allows an inductive signal to be read by the responding cell. When this signal is read, then the cell knows what type of other cells to make.

• Competence(Bias(Specified(Determined(Differentiation -Paracrine vs Juxtacrine: • Paracrine: Inducing molecules o Secreted factors o Soluble proteins secreted by inducing cells Bind to cell membrane receptors in competent cells o Exert their effect through signal transduction pathways -Major paracrine factors: 1. FGF 2. Shh/Hedgehog 3. Wnt 4. TGF-B-superfamily (BMP) • Wnt Signaling: o Inhibits GSK-3 o Allows B-catenin to localize to the nucleus and act a s a transcription factor o Activation by inhibiting and inhibitor o Wnt(frizzled(disheveled–]Gsk-3–]B-catenin(transcription Chapter 4 -Fertilization-what does it achieve and what are the main events during conception? • Fusion of gametes o Sex(variation)-transmission of genes o Reproduction-initiation of development Events of conception: 1. Sperm-egg contact 2.

Sperm entry regulation 3. Fusion of pronuclei 4. Metabolic activation of development -Spermatogenesis and oogenesis: COME BACK! -Meiotic arrest in and surrounding layers of an oocyte: • Oocyte: unequal cytokinesis to make polar bodies • Different species arrest at different stages: o Pre-meiosis o In Meiosis I o In Meiosis II o Meiosis Complete • Egg layers: o Cortical cytoplasm ? Cytoskeletal elements ? Cortical granules o Plasma membrane and associated layers ? Vitelline envelope-very tightly clinging ? Zona pellucid; corona radiate o Egg jelly -Sperm attraction: Chemoattraction of sperm to egg • Exocytosis of acrosomal vesicle • Sperm binding to egg membrane • Spernt entry • Fusion of egg and sperm membranes • Attraction and species-specificity: (Sea Urchin) o Ph of seawater or semen facilitates sperm motility o Resact-protein made by egg jelly helps attract sperm and keep alive o Bindin-protein on the acrosome, recognized egg cell membrane • Acrosome rxn: 1. Release of enzymes ? initiated by egg jelly contact 2. Extension of acrosomal process ? Tethers sperm to egg surface 3.

Formation of fertilization cone • Blocks to polyspermy: o Fast: Na-K pump Egg membrane has -70mV resting potential ? Influx of Na+ causes rise in potential ? Inhibits binding of other sperm o Slow:Ca2+ ? Cortical granule exocytosis ? Fusion with membrane causes elevation of vitellin to “fertilization envelope” ? Internal Ca2+ ion concentration is key • Male pronucleus entry, completion of female pronucleus meiosis, fusion of pronuclei, initiation of mitosis and metabolic activation of the egg???? Chapter 5 -Early development events: • Cleavage-cytoplasm divided, stays the same size o Cleavage-stage cells=blastomores • Gastrulation-multilayered body plan is established Cells that form the endodermal and mesodermal organs are brought to the inside of the embryo while ectodermal cells are spread over its outside surface • Cell specification and axis formation-cell fates specified by cell-cell singaling or by asymmetric distribution of patterning molecules into particular cells. -MPF (mitosis promoting factor) • Most important event in transition o Major factor responsible for the resumption of meiotic cell divisions in the ovulated frog cell. o Continues to regulate the cell cycle of early blastomeres after fertilization. o Activity highest during M and undetectable during S. Shift between M(S due to loss and gain of MPF • Consists of 2 subunits: (causes the cyclical activity) o Cylin B= larger subunit ? Accumulated during S and degraded after cells reached M ? Encoded by mRNAs in the oocyte cytoplasm, if the translation of message is specifically inhibited, cell will not enter mitosis. ? Regulates the small subunit of MPF o Cyclin Dependent Kinase=small subunit ? Activates mitosis by phosphorylating several target proteins.

? Brings about chromatin condensation, nuclear envelope depolymerization, and organization of mitotic spindle • Without cyclin B by cdc2 of MPF will not function. Cell cycle during cleavage: M S (No G1 or G2) • Driven by cyclin synthesis and degradation. -Cell cycle after the MBT: • Addition of G1 and G2 • Zygotic transcription ends (maternal components used up) -Patterns of cleavage: • Holoblastic=cleavage furrow extends through entire egg. o Isolecithal-eggs have sparse equally distributed yolk. ? Radial-sea urchin ? Spiral-snail ? Bilateral-tunicates ? Rotational-mammals, nematodes o Mesolecithal-moderate yolk ? Displaced radial-amphibians • Meroblastic=only a portion of the cytoplasm is cleaved, cleavage furrow does not penetrate the yolky part of cytoplasm. Telolecithal-dense yolk throughout most of cell ? Bilateral cleavage-cephalopod molluscs ? Discoidal cleavage-fish,birds o Centrolecithal-yolk in center of eg ? Superficial cleavage-most insects -Gastrulation: • Blastomeres undergo dramatic movements and change their positions relative to one another.

• Very important because it establishes three primary germ layers • Archenteron-future digestive tract • Blastopore-??? -Sea Urchin: • Cleavage-radial holoblastic • 4th cleavage begins to divide the embryo into mesomeres, macromores, micromeres. These tiers will adopt different fates (fate is reversible) • Blastula formation o All cells are same size o Ciliated blastula rotates w/in the fertilization envelope • Regulative development o Tiers and cell-cell interaction determine fate (micromeres) ? Exp: remove micromeres from embryo and place on top of isolated animal cap, animal cap will generate endoderm • Autonomous-inherit maternal determinants that had been deposited at vegetal pole • Conditional (regulative)-micromeres produce paracrine and juxtacrine factors that specify fates of their neighbors. Axis specification- o General cell fates line up along the A-V axis established in the egg cytoplasm prior to fertilization. o Animal-vegetal also structures the future A-P axis o D-V and L-R specified after fertilization o Nodal establishes R-L ? Exp: cloned nodal in situ demonstrated Nodal protein becomes expressed in oral ect. At 60. • Gastrulation-blastula consists of a single layer of about 1,000 cells that form a hollow ball o Ingression of the skeletogenic mesenchyme o Invagination of the archenteron o Convergent and extension -Snail: • Cleavage-sprial holoblastic Cells touch each other at more places than those of radial o Undergo further divisions before begin gastrulation o No blastocoel • Axis formation-polar lobe o Mosaic development-blastomores are specified atunomously o Cytoplasmic localization-morphogenetic determinants are placed in a specific region of the oocyte.

o Patterning molecules appear to be bound to a certain region of the egg that will for the polar lobe. ? Exp: remove polar lobe from trefoil stage, remaining cells divide normally instead of normal trocophore larva, result is incomplete larva.

Lacking endoderm and mesodermal organs as well as ectodermal. o Polar lobe contains the endodermal and mesodermal, these determinants give the D blastomore its endomesoderm forming capacity. o Localization of the mesodermal determinant is established shortly after fertilization, demonstration that a specific cyptoplasmic region of the egg, destined for inclusion in the D blastomere.

• Gastrulation-epiboly o Micromeres at animal multiply and overgorw the vegetal macromeres. o Micromeres cover entire embryo leaving a small blastopore slit at vegetal pole. -Tunicates: • Cleavage- bilateral holoblastic cleavage First cleavage plane establishes the earliest axis of symmetry seperating future right and left. o Mirror image cleavages o 2nd cleavage creates two large anterior cells • Most blastomores specified autonomously each cell acquiring a specific type of cytoplasm that will determine its fate. • Cytoplasmic loacalization: o Unfertilized=central gray cytoplasm enveloped by a cortical layer containing yellow lipid inclusions.

o Meiosis=clear accumulates in animal ? Clear and yellow(vegetal. o Yellow lipids migrate with sperm pronucleus(yellow crescent from vegetal to equator. o Produces tail muscles. Clear-ectoderm o Yellow-mesoderm o Slate-endoderm o Light gray-neural tube and notochord o Cytoplasmic-totaion with male pronucleus migration move yellow cytoplasm to final position. • Molecules involved: o Macho-1=activates muscles o FGF=activates cascades that block muscle formation • Gastrulation- o Invagination of endoderm o Involution of mesoderm o Epiboly of ectoderm o Convergence and extension of the mesodermal cells. Allows formation of notochord.

-C. Elegans-nematodes • Cleavage-rotational holoblastic o Produces founder and stem cell • Axis early specified o A-P earliest o Sperm entry=posterior D-V in division of AB cell o L-R determined later • Autonomous and conditional o Separate 1st two blastomores ? P1=autonomous without AB ? AB=conditional • Gastrulation- o Begins early o Cell ingression and migration to interior followed by epiboly o Migration continues and forms organs Chapter 7 -Amphibian: • Cleavage-displaced radial holoblastic • 1st 12 cell cycles roughly synchronous • No G1 or G2 • Morula 16-24 cells • Blastula 128 cells • MBT o About 4000 cells o Zygotic transcription activated o Differential transcription begins in different cells o Blastomores gain motility Axes symmetry- o Animal-Vegetal axis o Dorsal-Ventral axis: sperm entry point marks future ventral side o Following fertilization, cortical cytoplasm rotates ? Visible in some species as the “grey crescent” ? Maks the spot where gastrulation will being ? Rotation occurs on microtubule tracks • Gastrulation- o Vegetal rotation and invagination of bottle cells o Involution at blastopore lip o Convergent extension of the dorsal mesoderm -Mechanism of gastrulation: • Cadherin expression • Calcium ion surges • Fibronectin Transplantation of dorsal blastopore lip-Spemann organizer: • Newt blasomeres have identical nuclei each capable of production and entire larva • When separated by D-V different result • When 2 blastomores separated such that only one cell contains gray crescent=only gray crecent blastomore forms normally. • Conditional- cells of early gastrula=uncommitted • Autonomous-late gastrula=committed • Only dorsal lip of blastopore had autonomous fate • Referred to dorsal lip cells and their derivatives as organizer bc o Induced the host’s ventral tissue to change their fates to form a neural tube and dorsal mesodermal tissue. Organized host and donor tissues into a secondary embryo with clear A-P and D-V axis • Progeny of dorsal lip cells induce the dorsal axis and the neural tube is called the primary embryonic induction. • Cells in right place at right time form organizer. o 1st signal tells them they are dorsal o 2nd tells them they are mexoderm -What does the organizer do? What tissue does it become? What molecules important in NC? • Organizes dorsal side • Becomes dorsal mesoderm • Molecules=noggin follistatin -Nieuwkoop center-in the endoderm; it induces the Spemann organizer; what are molecules? Molecules=B-catenin • Becomes endoderm • Important-signals organizer -S.

O. found on dorsal vegetal above N. C N. C. found dorsal vegetal below S.

O. -Neural induction: • BMPs in ventral (ectoderm will become epidermis) vs blocking of BMPs by organizer molecules (noggin, chordin, follistatin) in dorsal (ectoderm will become neural tissue). • Epidermis is induced to form, not the neural tissue • Ectoderm is induced to become epidermal tissue by bindin BMPs, while the nervous system forms from that region of the ectoderm that is protected from epidermal induction by BMP-inhibiting molecules. o Default ate of ectoderm is to become neural tissue o Certain parts of the embryo induce the ectoderm to become epidermal tissue by secreting BMPs o The organizer tissue acts by secreting molecules that block BMPs allowing the ectoderm “protected” by these BMP inhibitors to become neural tissues: ? Noggin ? Chordin ? Follistatin • IGFs required for formation of anterior neural tube -Neural patterning (A vs P) by Wnt: • Head inducer(Wnt • Induce head structures by blocking the Wnt pathway as well as by blocking BMP 4 o Inhibitors: ? Cerberus ? Frzb ? Dickkopf -Fish: Cleavage-teleolecithal (most of cell is yolk) o Cleavage can occur in the blastodisc=yolk free cytoplasm at animal pole. o Meroblastic (discoidal) o 10 rapid cleavage cycles o Cleavages produce a blastoderm • Axis Specification- o Anterior-Posterior ? Established by enzymatic degradation of RA in the anterior end of embryo • Cyp26 ? Repressed by Wnt in the posterior o Left-Right Axis ? Very pronounced ? Clockwise rotational movement of a cilia patch( resulting in leftward flow of particles ? Ca2+ ions stimulate left-side specific pathways • Gastrulation- o Epiboly=blastoderm cells cover yolk Yolk mass is enveloped completely ? Blastoderm thickening as epiboly occurs: the germ ring • Outer layer: epiblast • Inner layer: hypoblast (Both thicken on dorsal side to form the embryonic shield) • Has axis organization properties -Unique features: cleavage, YSL, epiboly, embryonic shield (fish organizer) • MBT o Cleavages slow, zygotic transcription initiates, cell movement begins • 3 cell populations evident: 1.

Yolk syncitial layer (YSL) 2. Enveloping layer (EVL) 3. Deep cells-these will give rise to the embryo proper • YSL cells induce embryonic shield. Epiboly(germ ring • YSL cells on dorsal side accumulate B-catenin(unduce embryonic shield. o Turn on production of squint and Bozozok ? Bozozok represses BMP and Wnt activity ? Blocks inhibition of organizer genes ? Activiates chordin, noggin, follistatin, dickkopf o Together B-catenin and Nodal-related 1 specify the dorsal side of the embryo. Chapter 8 -Birds and mammals- • Cleavage- o Birds-meroblastic (small portion of egg cytoplasm atop yolk mass undergo cleavage) o Mammals-holoblastic rotational (cleave modified to form placenta) • Mode of gastrulation- Bird-epiblast and hypoblast formation, primitive streak, primitive groove, Hensen’s node o Mammalian- gastrualation and implantation, epiblast and hypoblast (bilaminar germ disc), primitive streak • Axis specification- o Bird- ? Gravitational influence ? Cells of posterior marginal zone= N.

C. o Mammalian ? Primitive streak= P-A ? Node forms at posterior end ? AVE -Unique features of chick development: • Rapid development (3 weeks) • Internal fertilization prior to albumen and egg shell secretion • Amniotic egg=amniotic sac, chorion-help get oxygen; way of ensuring development can take place on land. Blastodisc-flat area on top of yolk where development takes place Cleavage-meroblastic discoidal • Telolecithal with blastodisc atop yolk mass • Initially syncytial blastoderm later divides into multiple layers • Subgerminal cavity forms b/w blastoderm. • Area pellucida: center of blastoderm • Area opaca: edges of blastoderm -Chick gastrulation- • Pre-Gastrulation- o Cells of area pellucid at surface: form epiblast o Some cell delaminate, form primary hypoblast in the subgerminal cavity. o A sheet of cells migrate P to A and pushes hypoblast anteriorly. o Result: a 2 layered embryo.

Epiblast and hypoblast ? Joined at 2 edges, these 2 layers separate and form a blastocoels • Gastrulation- o Primitive streak forms ? Cells arise in posterior marginal zone ? Thickening of the epiblast ? Cells thicken and undergo convergent extension ? Streak cells extend and progress anteriorly o As the streak forms, also creates a primitive groove. ? Cells migrate through groove and into blastocoels o Hensens’s Node: thickening or “primitive knot” at the leading anterior edge of the streak. ? A place where cells can migrate through to the blastocoels ?

Functionally equivalent to the organizer (frog) and the shield (fish) o Through the primitive streak/ Hensen’s node, epiblast cells stream through the streak (ingress): 1. Endodermal precursors move inside first 2. Hensen’s node forms notochord and anterior somites 3.

Other ingressing cells form somites, heart, kidney, and other mesoderm. 4. As the streak moves forward, ingreassing cells move to take up proper position. o Hypoblast cells help guide the primitive streak anteriorly. • As it progresses: Ingressing endodermal cells push hypoblast cells off to the side.

o All endodermal organs plus most extraembryonic membranes form these endodermal precursors. o Middle layer of cells are loose and migratory and will give rise to the mesodermal structures. • Completion: o Regression of the primitive streak o Hensen’s node now pulls back (posteriorly) and leaves notochord in the wake o Node regresses all the way to the caudal/ anal region o All cells remaining on the outside of the epiblast will adopt ectodermal fate. o All axes now established o Epiboly proceeds to spread ectoderm around yolk. Primitive streak moves P to A; in regression goes back A to P: • Moves posterior to anterior (creates groove) • Regression-moves anterior to posterior (node regresses) • Axis formation: o A-P:gravitational influence o As egg passes through oviduct, it spins o Spinning causes lighter comp. to lie upward o Lighter region is posterior side of blastoderm • Similarities: o Hensen’s Node=organizer o Cells of posterior marginal zone=N.

C. ? Capable of forming primitive streak and hensen’s node o Transplantation of H. N. to another site causes formation of second axis. -Unique mammalian features: Small embryos • Rotational cleavage • Internal fertliziation o In ampulla of oviduct o Sperm contact triggers completion of Meiosis II • Little yolk • Features of early development: o Compaction about 8 cell stage o Formation of blastocyst: • 2 parts distinct by 64 cell stage ? Trophoblast=chorion ? ICM=embryo proper and other extraembryonic membranes.

-Blastocyst- • Blastocyst moves toward uterus during cleavage and formation of trophoblast and ICM; also sheds or hatches form zona pellucida • While movement of embryo to uterus through oviduct occurs o Shedding of zona pellucid (follicle cells) ? Z. P. revents adhesion and ectopic pregnancy ? Shed upon arrival at the uterus (“hatching”) ? Allows implantation -ES cells, totipotency, pluripotency: • Blastomores are totipotent to 8- cell stage and beyond • ICM cells are pluripotent • Specific transcription factors in ICM –maintain pluripotency and allow ICM cells to remain as ES cells. -Gastrulation and implantation: • Occurs simultaneously • ICM splits into hypoblast and epiblast. o Hypoblast-delaminates to line blastocoels and from yolk sac o Epiblast-will give rise to amnion and embryo proper • Gastrulation similar to chick Node forms at posterior end and primitive streak moves P to A.

o Cells stream through primitive groove into space between epiblast and hypoblast o Hypoblast pushed out of way by endoderm • Cell arrangements prior to gastrulation: o “bilaminar germ disc”-epiblast and hypoblast o Hypoblast cells pull away to from yolk sac o Epiblast cell layer splits to form amniotic layer and embryonic layer o Amniotic cavity begins filling with amniotic fluid -Where does placenta come from? • Formed from deciduas (maternal component) and chorion (embryonic component) • Trophoblast splits into cytotrophoblast and syncytiotrophoblast. Cytotrophoblast-adheres to endometrium, integrates with maternal blood supply, forms chronic villi o Syncytiotrophoblast-digests uterine tissue to facilitate implantation. Forms umbilical cord, blood vessels. • Endometrium (uterine lining) modified for blood supply to form chorio. • Nutrients, oxygen, CO2, and wastes diffuse across the villi. -Categories of twins: • Monozygotic-idnetical • Dizygotic-fraternal • Monozygotic twin/ multiple formation: o Speration fo blastomeres early o Separation of ICM at blastocytes stage (later) When split occurs, determines extent of shared membranes: o Before day 5 (before trophoblast formation, before amnion formation): two separate chorions and amnions.

o B/w days 5 and 9 (after trophoblast formation, before amnion formation): shared chorion, but two separate amnions o After day 9: shared amnion ? Twins may develop as conjoined • Axis formation o Mouse embryo cup shaped, not flat o A-P axis: ? Anterior signaling from the node and from the anterior-most endoderm (AVE) o AVE ? Nodal (in epiblast) activates posterior mesoderm genes. Nodal antagonized by Cerberus and Lefty1 expressed in AVE(allow anterior expression ? Ave expresses anterior genes including Wnt inhibitors • Suppresses posterior patterning o Node ? Node arises in the posterior and secretes chordin and noggin as it moves P to A. ? N and C act to antagonize BMP (ventral patterning) • If n and C are both knocked out, no forebrain or face will form. o Molecues for P: ? BMP ? FGF-pattern cdx gene expression (posterior) which activates some Hox genes ? Wnt Retinoic acid- anterior (degrading) and posterior (synthesizing) enzymes set up gradient. ? A-P polarity ultimately determined by Hox gene expressions -Hox gene cluster • Mammals have four copies of the homeotic gene complex.

1. Hoxa 2. Hoxb 3. Hoxc 4. Hoxd • Order of genes conserved from fly to human with regard to o Chromosomal position A-P expression pattern. o Mammalian Hox genes are homologous to fly homeotic genes o Similar genes b/w each set of mammalian Hox genes are paralogues ? Gene duplication o Head patterning: non-Hox genes ? Orthodentricle and empty spiracles in fly ?

Otx and Emx in mammals.

• Body segment identity A-P gained from posterior most Hox gene expressed there o And combined expression of paralogues • Knockout of all Hox paralogues of a certain level leads to lack of vertebrae characteristic of that level o Transformation identity. • Misexpression of Hox genes (and alteration of segment identity) can be caused by retinoic acid. • Hox gene expression determines type of vertebrae formed Part III Chapter 9 -Stem cells: • Embryonic: from ICM • Adult: in mature organs: retain potency; replace and repair a subset of cell types • Potency Totipotent (zygote) make anything o Pluripotent (embryonic stem cell) make almost anything o Multipotent (multipotent stem cell) o Restricted o Terminally differentiated • Stem cell=undifferentiated cell that divides to produce: o A cell that retains its undifferentiated character o Acell that can undergo one or more paths of differentiation • Can renew themselves at each division while producing specialized cells • Cell replacement • Repair -Fate of the ectoderm • Neural vs Epidermal precursor cells • Neural specification occurs stepwise • Neural precursor cells (neuroblasts) o Competence Specification o Commitment/determination o Differentiation • Ectoderm: o Surface ectoderm-epidermis, nails, hair o Neural crest-PNS, adrenal medulla, dentine of teeth o Neural tube-brain, spinal cord, motor neurons, retina -Neurulation-closes like a zipper • Neural plate has been induced o Inhibition of BMPs by organizer genes o Neural plate has been regionalized A-P ? Wnt, FGF, RA in posterior have turned on Hox gene expression ? Inhibition of posterior signals allows anterior patterning o Patterning and secondary neurulation occur to close the neural tube • Ways to form neural tube: Primary neurulation (anterior neural tube) ? Hollow tube pinches off ? Neural tube becomes internalized (brain and spinal cord) ? Epidermis remains outside ? Neural crest cells wind up in b/w (PNS, glia, pigment cells) o Secondary neurulation (sacral neural tube, tail) ? Solid cord arise and hollows out • Human steps in primary neurulaton: o Neural plate formation ? Notochord signals overlying cells to elongate/thicken o Neural plate shaping ? Convergent extension/lengthening of plate. o Neural plate bending to form neural groove. ? Cells at hinge regions become wedge-shaped ?

Neural folds rise up o Neural tube closure ? Folds adhere and cells from both sides merge • Neural tube closure-TIMING o Chick, mammal have long body axis o Neural tube closure occurs in stages, not all together o Chick- ? Cephalic neural tube closes before caudal ? Closure starts in midbrain and zips up in 2 directions o Mammal- ? Multiple sites for closure ? Posterior neuropore failure: spina bifida ? Anterior neurupore failure: anencephaly ? Entire neural tube non-closure: craniorachismisis • Important molecules o Cell adhesion molecules ? N-cadherin, N-CAM Dietary factors ? Folate (folic acid, vitamin B-12) • Folate receptor proteins prior to closure ? Cholesterol • D-V patterning o Dorsal spinal cord: sensory neuron input o Ventral spinal cord: motor neuron output o Between: interneurons -Source of D-V: • Ventral spinal cord patterned by notochord: Shh • Dorsal spinal cord induced by epidermis: TGF-B family -D-V patterning in neural tube: • Shh induces groove/hinge cells to become floor plate of neural tube. o Floor plate secretes shh and forms a gradient V to D • TGF-B family members (BMPs) in epidermis induce dorsal cells to become roof plate.

Roof plate secretes BMP 4 and induces a cascade of related factors D to V. • Hox code (A-P) combine with this D-V patterning specifies all the neurons’ identities. -White Matter vs Gray Matter • Gray-neuron cell bodies, nuclei (cortex) in CNS, ganglia in PNS • White-axons, tracts in CNS, nerves in PNS -Neuron birthday and migration to form distinctive brain regions: • Originally neural tube is one cell layer thick (germinal neuroepithelium) o Actively dividing neural stem cells o Continous from lumen to outside • When cell begins to differentiate into neurons, a stem cell divides. Progeny migrates and (terminally) differentiates o The neurons “birthdays” o Those cells born the latest migrate to the cortex • Cells near lumen continue dividing (germinal neuroepithelium, later called ventricular zone) • Outer layer (where cells have migrated) is the mantle or intermediate zone • Axons are sent further forth creating a marginal zone. -Vertebrate eye development- • Transcription factors including pax6 and Rx1 are expressed in the anterior most neural plate. • Pax6 is a master eye regulator (smalleye, aniridia) • The domain of expression is split (inhibited by) Shh.

Loss of shh: cyclopia o Gain of function Shh: lack of eye formation Chapter 12 -Fate of the lateral mesoderm: • Lateral plate mesoderm and endoderm interact: o Circulatory system o Digestive system • Lateral plate mesoderm splits into 2 layers: o Dorsal: somatic mesoderm o Ventral Splanchnic mesoderm • (Space in b/w becomes the coelem) -Heart development: • Migration of cardia primordial: o Heart precursor cells migrate along fibronectin and stop near the gut. o As gut folds inward, L and R cardiogenic mesoderm brought together ?

Severing of this tissue to prevent L&R mesoderm from meeting= cardia bifida (2 hearts) • Heart looping: o Moves atria to an anterior position o Establishes L&R polarity ? Requires L-R polarity genes Nodal, Lefty ? Differential cells proliferate as heart grow and expands ? Formation of functional valves o Chambers: ? High RA in posterior specifies venous fate; atria ? Fused hear tube develops vitelline veins (leads to sinus venosus and atria), truncus arteriosus (aorta) -Fetal to Newborn: • Fetal= mix of oxygenated and deoxygenated blood • Fetal: Foramen ovale is open b/w atria o Ductus arteriosus is open • Newborn o Ductus arteriosus closed o Foramen ovale closed Chapter 13 -Limb polarity and 3 axes: • Cells b/w arm and leg common • L and R not same • Pattern is common Axes: • Proximal-Distal (shoulder-finger) • Anterior-Posterior (thumb-pinkie) • Dorsal-Ventral (knuckles-palm) -Limb bones: • Stylopod-attaches to axial skeleton o Humerus, femur • Zeugopod-medial bone o Radius/ ulna, tibia/ fibula • Autopod-most distal o Carpals, tarsals -Molecules in patterning: • Prox-dist: FGF • Ant-Post: Shh (Shh=pinkie, no Shh= thumb) • Dors-Vent: Wnt Limb bud formation: • Limb field- all cells in an area capable of forming a limg o Mesenchyme (loose medoerm) proliferates under the ectoderm o This creates a bulge: the limb bud (core of mesoderm overlaid by epidermis) • Buds elongate and flatten o Muscles and cartilages differentiate in proximal to distal direction • Hox gene expression is key in determining where limb buds will form o Forelimb formed at anterior limit of Hoxc6 (first thoracic vertebra • Epidermis thickens to form AER o Apical ectodermal ridge o Epidermis will form scales, claws, etc. Limb bud mesenchyme forms all skeletal structures of limb o Cartilage, tendons, ligaments, dermis • Muscle fibers come from nearby somites. • FGF10 signaling form lateral mesoderm sets up reciprocal interaction b/w mesoderm and ectoderm o An implanted FGF bead will iduce a new limb bud o FGF 10 K-O: no limb buds form o FGF 10 expression restricted to fore/hindlimb regions by Wnt genes. • AER secretes FGF8, keeps mesenchyme proliferating o Progress zone • If AER removed, limbs truncated • Underlying mesenchyme expressing Shh o The ZPA (zone of polarizing activity) -Role of ZPA in limb outgrowth: ZPA is in posterior mesenchyme of limb bud o Responsible for A-P patterning of limb • Grafting a ZPA (or the Shh bead) to the anterior limb produces a double limb • ZPA established by reciprocal interaction b/w FGF in ectoderm with mesenchyme.

• Limb forms prox-dist (stylopod, zeugopod, autopod) • Hox induced by Shh o K-O of Hox can cause no zeugopod to form -Digit identity: • Thumb most anterior, pinkie most posterior • Shh expressed in ZPA • Become bone, muscle and skin of posterior limb. o Pinky,ring, half of middle • The amount of time Shh is expressed specifies digit 5 from 4 from 3. o Secreted Shh specifies digit 2 Digit 1 is specified by absence of Shh -Role of BMP in formation of separate digits: • Shh may be working through BMPs • Apoptosis responsible for joint formation and separation of digits • Mediate by BMP and BMP antagonist Gremlin o By inhibiting BMP, Gremlin allows webbing to persist b/w digits o BMP induces apoptosis and promotes cartilage Chapter 15 -Wnt(necessity in regeneration: • Blocking Wnt signaling through B-catenin blocks regeneration in fish tails. o Enhancing B-catenin(enhances regeneration o Wnt necessary for regeneration (seen in liver regeneration in mice) -Regeneration: 4 types: o Stem-Cell Mediated: ? Hair growth, blood cell o Epimorphis: ? Adult structures dedifferentiate, proliferate, and become respecified ? Planaria, salamander limbs o Morpholaxis: ? Repatterning of existing tissue with little new growth. ? Hydra o Compensatory ? Dedifferentiated cells divide and produce more cells of the same type ? Mammalian liver regeneration.

• Epimorphis: o Missing structures regenerated o Prox-Dist identity clearly established in the cut edge o Involves: ? Cell dedifferentiation ? Cell proliferation ? Cell respecification • Morphallaxis: Budding in hydra ? No true mesoderm(dipoblast) o Can reproduce sexually, but usually do so asexually. o Extensive cell division, migration and shedding. o Cut hydra, get smaller hydras o Cell retains plasticity o Can also homogenize • Compnesatory: o Mammalian liver can accomplish. o Differentiated cells divide and recover structure and function of an organ. o Remaining lobes enlarge to compensate for lobes removed. o Cells become mitotic without any dedifferentiation o Feedback regulation of regeneration from: ? Growth factors, insulin, thyroid hormone, norepinephrine -Regenerating Human Limbs: Salamander: (only vertebrate able to regrow complex body parts) o Cut limb o Forms apical ectodermal cap o Dermis does not cover over and move with the dermis • Underlying cells dedifferentiate and detach o Forms blastema o Cell-specific genes turn off and genes associated with lilmb outgrowth are activated- these cells have no identity o FGF molecules involved • After amputation: o Limited bleeding, no scar o AEC forms o Blastema froms • What is needed? – o Rerouting a nerve to a wound site forms a blastema o Fibroblasts from opposite site of limb needed.

(role of an FGF circuit) • Positional info is key Adult salamander fibroblasts maintain positional Hox code info access • Crosstalk b/w fibroblasts • Muscle “dedifferentiate” is actually the activation of muscle steam cells • Developing genes reactivated in epidermis o FGF 10 and Wnt 7a • BMP 4 needed for regeneration Humans: • Fingertip: o Cean and dress wound, do not suture a skin flap over wound (prevents AEC from forming) • Dermis is a problem (doesn’t regenerate) o Rich in fibroblasts o Undergo fibrosis (healing and scar tissue formation) o How to stop fibroblasts from forming scar tissue? ———————– Covert Overt