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uploaded by user Yuyumon Class: Lecture/Exam: School: Semester: BIO 325 Exam 2 SBU Fall 2012

Exam 2 - Guide

Differences between mammals and xenopus:

A.events set up for gastrulation unique to mammals (internal) B.Cell cleavage, mammal rotational, xenopus horizontal

Most studied animal Cell definitions Blastomere Totipotent

Mouse, most studies concern murine development

Cells produced by cleavage of zygote Earliest blastomeres, up to 8 cell stage. Can form trophoblast cells and embryo precursor cells ICM, cannot form trophoblasts. Can form any other cell in body. Factors Oct4, Stat 3, Sox2, Nanog maintain pluripotency Undermethylated, DNA strands open and allow for transcription of a lot of things. Once they differentiate and specify they become more methylated.

Pluripotent

Zona pellucida Extracellular matrix of egg that was for sperm binding during fertilization Prevents blastocyst from adhering to oviduct walls Once it reaches uterus blastocyst attaches to stick to wall Glycogen layer, monocellular, made by egg. Zygote hatches out of it. Proteins on surface that sperm recognizes, so that sperm can penetrate Polar body Zygote ICM Blastocyst Diploid cell resulting from fusion of haploid sperm and egg makes embryo, yolk sac, allantois and amnion Preimplantation embryo, blastocoel, focal cluster (ICM), peripheral TE (future placenta) ICM is made up of Epiblast (future entire embryo) and Hypoblast (primitive endoderm) for future yolk and nutrients Chorion Enables fetus to get oxygen and nutrients from mother, fetal portion of placenta Secretes hormones that cause uterus to keep it Secretes regulators of immune response so mothers immune system doesnt reject it Made from external cells of morulla (trophoblasts)+ mesoderm cells from ICM

Decidua

Maternal portion of placenta, rich in blood vessels

Morulla Totipotent cells Hypoblasts Epiblast lower layer of ICM, primitive/visceral endoderm (express gata6 transcription factors) Upper layer of ICM, epithelial tissue, expresses nanog transcription factor Gives rise to all three tissue layers of embryo Bilaminar germ disc Trophoectod. Structure of blasts and epiblasts

Outer layer of blastocyst stage embryo Made from first differentiation event in mammalian development Adheres to uterus lining Digest wall of uterus to go deeper into uterus Gives rise to extraembryonic ectoderm after implantation and make embryonic part of placenta

E/abemb. Axis Axis defining if cell differentiate to TE or ICM Side of ICM in blastocyst is embryonic, cavity (blastocoel) side is abembryonic Endometrium Epithelial lining of uterus Made from sugars, collagen, laminin, fibronectin, cadherin, hyaluronic acid and heparin sulfate receptors Extraembrionic endoderm Hypoblasts delaminating from ICM to line blastocoel cavity Forms yolk sac Embryonic epiblast from epiblast, all cells that generate actual embryo -> similar to avian epiblast

Amniotic cavity Epiblast, filled with amniotic fluid, shock absorber, prevents drying out Teratogens Exogenous agents causing birth defects Mostly affect embryo during embryonic period to end of week 8 (max susceptibility) AND fetal period Chromatin remodeling Change in structural properties that affect accessibility to protein factors trying to express gene RNA induced silencing Complex that inhibits translation of target RNAs through degradation

Mater SCMC

part of subcortical maternal complex (SCMC), on apical surface of cells Maternal gene products, asymmetrically localized on apical side within blastomeres, made from Flopped, TLE6, Filia, Mater Accumulate in egg, increase till ovulation and persist, but decrease linearly until 2 cell stage were not present anymore No SCMC in ICM

Extraeembr endoderm Form yolk sac, hypoblasts delaminating from ICM to line blastocoel cavity Embryonic epiblast Form epiblast, all cells that generate actual embryo

Amnionic cavity From epiblast, filled with amniotic fluid, shock absorb and prevents drying out.

Cell development

Mammilian Development Fate of eggs Successful fertilization 17/20 Implantation 14/20 Gastrulation to 4th week 8/20

Coming to term 6/20 Infant mortality In high developed country 30% of kids die of birth defects -> something must happen in development Birth defect A. Chromosomal abnormalities (leading cause) B. Environmental defects C. Multifactorial interaction between genetic composition and environment Defect Syndrome If multiple organs affected it is called syndrome If due to single gene -> pleiotropy Can also be due to many genes Single gene defect Mosaic Tissue affected by gene is in multiple organs. Same gene used in many types of tissue. If gene defective, it affects many organs at once Cascading effect of one defective gene in one cell. Doesnt function correctly and affects correct functioning of second organ.

Relational

Genetic heterogeneity Genetic disorder might be the result of any one of many gene alterations. Ex.: If signal transduction pathway is non-functional, it could be any of the components in signal transduction pathway that is broken. Phenotypic heterogeneity Gene defect can result in a variation of phenotypic effects. This is due to the fact that genes are not autonomous, but rather influenced by other gene products and environment. Ex.: Diabetic people can fall over, or go into shock, depending on situation. Mouse cycle 4 days 4th day 6 days 10 days 10-14 days Final days Day 20 Total 6-8 weeks Cleavage differentiated vs. pluripotent cells Implantation Epiblast, epithelial layer all organ rudiments present further differentiation Maturation Birth, but development continues after birth

Why hard to study?

Smallest eggs in kingdom -> hard to manipulate Not produced in great number -> hard to obtain material Development inside body -> only till recently been able to simulate

Why cleavage diff.?

1. - Prior to fertilization oocyte wrapped in cumulus cells released from ovary and swept by fimbriae into oviduct Fertilization in ampulla of oviduct (region close to ovary). Meiosis is at sperm entry. First cleavage a day (24h)later (amongst slowest of animals) - Travel: Cilia in oviduct push embryo toward uterus 2. asynchrony of cell division: Mammalian blastomeres dont all divide at the same time. Means cells do not increase exponentially. Cant clearly define 2,4,8 cell stage, because number of cells can be odd. Divide slower than xenopus 3. Genome activated during early cleavage. Rapid developing animals only activate later. 4. Nuclei, not oocyte cytoplasm produces proteins necessary for cleavage and development

Mammalian gastrulation

movements are similar to birds and reptiles

Adaptations

Funny because mammals almost no yolk. Doesnt need it to get nutrients 1st. nutrients from mom, not yolk 2nd. Restructuring of maternal anatomy 3rd. chorion, fetal organ that can absorb nutrients

Rules of Evidence Correlative Evidence Find it, correlation between two events. Ex. Gene x is expressed just preceding event Y. Lose it, loss of function evidence but does not exclude other possibilities. Ex.: Knock out gene for signal transduction pathway -> product is gone Move it, gain of function. Demonstrates X causes Y, but could also be due to indirect stuff.

Functional Evidence

Functional Evidence

Model systems Life cycle Study multiple generations in short amount of time. Mouse ovulates every 4 days, gestates in 20 days

Meiosis Female oogenesis Meiosis once in finite amount of cells ONe gamete per meiosis Male spermatogenesis Meiosis constant, mitotically dividing cells Four gametes

Meiosis completion delayed for months Meioses completed in days or weeks Meiosis arrested No meiosis cell cycle arrest

Differentiation of gametes while diploid Differentiation of gametes while haploid, after meiosis All cells go through recombination Only autosomes go through recombination

Meiosis in oocyte Dictyate state Oocyte development in oogenesis stuck in meiosis prophase 1. Maintained by follicle granulosa cells. Release from follicle when spike in LH hormones. Green tubulin staining, blue chromosome staining Add luteinizing hormone to make egg developed, release from meiosis arrest Prophase 1 Metaphase 1 Anaphase 1 Metaphase 2 Nucleus breaks down Spindle migrates to lower part of cell from center Chromatin separates on metaphase plate One large cell, one polar body, which divides into two polar bodies Fertilization completes meiosis Pronuclei move from periphery to center. Cell becomes diploid zygote

Release Experiment

After fertilization Stages in development Division types

Differentiative (orthogonal) - Asymmetric divisions make ICM inside and TE outside. Perpendicular to polar axis of cell. Only one side gets SCMC Happens more often when CDx2 is down regulated, or at low levels Reinforces ICM production Conservative (parallel) Symmetric, both daughter cells remain TE and on

outside. Along axis of cell. Both get SCMC Happens more often when Cdx2 levels are high in cells. Why? Because it enhances apical localization of polarity markers such as PKC Reinforces making TE Cell divisions - First Fate Decision 8 cell stage 8-16 cells tage Cdx2 mRna on outside of cells Symmetric divisions at outside cells (cells not in center) make both cells inherit CDx2 mRNa Asymmetric division of cells make one center cell and one outside cell which inherits more Cdx2 mRNA than inside cell 16 cell stage All outside cells have more Cdx2 mRNA than inside cells

Animal pole

where meiotic division takes place -> makes polar bodies. Two polar bodies attached to each other mark axis. opposite to where meiotic division takes place Cell polarity: Asymmetric division localizes Cdx2 on apical side of cell. More Cdx2 makes more TE.

Vegetal pole Cell polarity

Pre Fertilization

Gametogenesis, Process of making gametes sperm and egg. Maternal products made in growing oocytes Gametes marking, mother and father individual marks. Maintained till next generation primordial germ cell development were removed.

Primordial germ cell

PGC, gamete progenitor cells Sex specific Imprint taken off each generation and new ones put on male cells, left off on female cells -> made into gametes that are now distinct.

Gametogenesis Sex marking

Process by which the sperm and egg are formed Imprinting: DNA modification, addition of a methyl group to cytosine followed

by guanine. Specific to oocyte or sperm. Imprinting always sex specific. Maintained during gametogenesis. Removed in primordial germ cells. DNA methylation either sex specific way, then called genomic imprinting, or genome wide, then non specific Addition of methyl group on cytosine Removed during zygote formation and then reestablished Epigenetic, can change each generation. Not nucleotide changes itself Impedes transcription of DNA when DNA methylated Fertilization zygote has high methylation, 2 cell stage has low methylation After blastocyst methylation increases again to high methylation during gastrula stage Embryos remethylate faster than extraembryonic tissue. Fertilization begins demethylation. Types of methylation Example: Maternal and Paternal Chr7, section with Igf2 and H19 genes and TF enhancer ordered one after the other on the DNA strand. CTGF attached in ICR between Igf2 and H19 gene. Igf2 effectively shut off, TF enhancer can only bend back and transcribe H19 Methylations in ICR between Igf2 and H19 gene. Igf2 on, because it is not blocked, TF enhancer can bend back and transcribe Igf2 and H19 Cytosine guanine dense. On paternal chromosome they have methyl groups, on maternal side they are unmethylated. ->CTGF attaches and makes brick wall. Protein on TGF enhancer cant bind to IGF2 and can only go to H19 Paternal methyl groups put on during gametogenesis Histone acetylation Histones are needed to wind or unwind DNA depending if it is needed for transcription. Each histone has a tail. These are made up of lysine amino acids that are positively charged and tightly wrap around the DNA to coil it up. To make the DNA available for transcription, histone tails are acetylated. Lysine charge is counteracted and DNA unwinds. Acetylation is an epigenetic tag on the histone.

Maternal chr7

Paternal chr7

ICR

Deacetylate it to make it coil up again and not usable for transcription. Histone tail becomes deacetylated as differentiation continues H3methylation high in ICM low in TE. Suppresses Cdx2 in ICM, enriched in TE due to activated histones (not methylated) Maternal pronucleus high levels of H3methylation, paternal pronucleus erased of methylation Both together cause change in chromatin structure (nucleosomes, DNA on Histone). During gametogenesis we go from open conformation where transcription is possible to a chromatin state where it is closed and transcription is impeded. At the end of gametogenesis gametes are transcriptionally impeded. During gametogenesis oocyte makes material and stores it, before it becomes inactive. Sperm much less so. After fertilization DNA needs to be reactivated. Ovary and oviduct not connected. Fimbriae draw eggs in. Fertilization in oviduct. Chromosome inactive in oocyte. Methylated and histones deacetylated. Fertilization Sperm recognizes proteins on zona pellucida and penetrates to fuse with egg. Triggers changes, activates zygotic genome, begins to transcribe. Day 1 2 pronuclei, Female and male pronucleus (gametic nucleus) begin to fuse. Polar body result of completion of meiosis 1, asymmetric cell division leads to it being smaller. Fertilization completes meiosis 2. Female pronucleus contains heterochromatin Plane or Axis of cell division evolves right between two pronuclei FIGLA Transcription factor needed for egg development. Regulates a couple of oocyte cell developmentally specific genes. Forms dimer with Tcf3. Both bind to enhancer element upstream of target gene Known to be needed for zona pellucida genes ZP1, ZP2 and ZP3 Loss of one copy of FIGLA in humans: Loss of egg production in 30s Loose both copies in mouse: Loss of egg production

Ovulation

Fertilized Egg

Experiment: Looked if other maternal genes are activated by figla using microarray to compare gene expression. Looked for RNA only present in wild type and not mutant -> Flopped was missing. FOUND: Flopped, mater, TLE6 and Filia missing (all part of SCMC) FLOPPED Essential for keeping mater localized, suggest its in complex maternal -/- x paternal -/+ = no viable offspring, mater is diffused Females are sterile, despite normal ovary and egg morphology. First cell divisions usually asymmetric and delayed. Aneuploidy and aberrant transcription Dont need 2 copies of Flopped for embryo to develop normally, only in mom. Flopped localized in egg on apical side overlapping actin, after cleavage is absent, it is absent from cell junction (where two eggs cleaved, so it remains only where the original apical membrane was of first cell) However, it is dynamic if cell contact lost in 2 cell stage, FLOPPED can relocalize around complete cell apical membrane again. Remains on apical membrane during blastocyst development (SCMC) Localization depends on where mater is, vice versa Mater localization exp. -/- flopped, mater protein is diffused and not localized Leads to aneuploidy and aberrant transcription Hippo signaling path Important for regulating size of organs Hippo pathway activated when Cdx2 not present Hippo could be involved in cell fate determination Components are conserved in vertebrates and functionally equivalent (YKI activator of transcription in drosophila, YAP in mammals) Contact activated signaling pathway. Apical signal suppresses hippo. Lack of cell polarity and contact activates hippo Chain event: If activated YKI/YAP phosphorylated and cannot go into nucleus. Genes for proliferation are off When hippo activated cells slow proliferation

If cell grown in dish there is no contact: Hippo off, YKI goes into nucleus onto SD in drosophila and activates cell cycle genes. Ex and Mer are scaffolding proteins Tead4 same as SD in drosophila Hippo mutations loss of function mutations (HPO,WTS,SAV) result in tissue overgrowth, hyperproliferation of cells. If happens in single organs they swell, tissue gets larger, because of less cell death. Mutant tissue proliferates faster. BrdU: Measure of DNA and direct measure of cell proliferation Incorporates thymidine analogue in DNA called BrdU. If cell is dividing it is incorporating BrdU. Cells that have lost hippo have large incorporation of BrdU Inactive Hippo pathway: YKI goes into nucleus. Cell proliferation increases, apoptosis slows down. Overexpression of HPO,EX or WTS: Reduce organ size, slow down proliferation, increases apoptosis Overexpression of YAP=YKI in mouse: Liver grows. Extended overexpression leads to hepatocellular carcinoma. YAPS168 mutation: Cannot phosphorylate or be ubiquitinated and goes into nucleus. Tissue overgrowth results from decrease cell death.

Demethylation

After fertilization DNA of gametes needs to open for transcription

 Reactivation through demethylation Male chromosomes become actively deactivated very fast (only global ones). Before first cleavage all genome wide CpGs (methylations) of male are removed -> activated. Male sex specific stay on. Female genome passively activated. After every cleavage demethylation only 50 %. New strands of DNA are not methylated, old ones left methylated. Female embryonic genome activated through cleavage Gradual loss of global demethylation. Zygotic transcription starts at low levels at 1 cell stage, but picks up to 100% at 2 cell stage. Completely activated! In mammals, take-over of zygotic factors very early, xenopus at gastrulation Factors Cleavage Maternal factors degraded by the RNA induced silencing complex. Cleavage every 24 hours in mouse, polar body cleaves first. Male nucleus depleted of epigenetic markers at time of first cleavage. Genome reactivated. Cleavage along animal/vegetal pole. Polar body at animal pole. First dividing blastomere has potential to divide into ICM & TE. Daughter cells stick together Mammal cleavage rotational. Result: Blastomeres have different maternal components. Animal pole has higher concentration of methyltransferase 1 (Carm1)Still have full potential to become anything or even two separate individuals. At 4 cell stage blastomeres have Av (usually go to center to become ICM), v, or A, potential to develop into certain things -> Distinct potential for development. Cells specified early on. Av cells 85% of chimeras make animal A cells 25% of chimeras make animal V cells 0% of chimeras make animal AV cells have higher levels of H3R17me21, vegetal material have lower of these modifications. Vegetal cells become less pluripotent contain more Cdx2, divide more symmetrically to make TE Compaction stage: Up to 8 cell stage, not yet determined, but capable of making anything. Real differentiation begins now.

1 cell stage

2 cell stage

4 cell stage.

At 8 cell stage mouse blastomeres are loosely arranged. After 3rd cleavage (after 8 cell) blastomeres express E-cadherin Blastomeres huddle together and form compact ball, look like epithelial cells. Ball stabilized by tight junctions, seal of sphere, inner cells sequestered. Small signal molecules and ions can pass in sphere Beginning of CDX2 expression. Differentiate division: Outer cells become TE, inner bipotential or epiblast. Conservative division: Always only 1 TE into 2 TE If trophoblast in zygotes is defective of Tead4 it cant go into uterus and expand. Leads to inability to implant because no Trophoblast creation Morulla stage: 16 cell embryo produced by 8 cell division. Small group of inner cells surrounded by large group of external cells. Internal cells called ICM and give rise to embryo. External cells will become trophoblast (throphoectoderm) cells. Form no embryonic structure. Form chorion, embryonic placenta. Whether TE or ICM is matter of chance, no subcellular components determine asymmetry as in xenopus. Plane of division number 1 completely irrelevant to emb/abembryonic axis FIRST DIFFERENTIATION: separation of ICM from TE Differentiate division: Outer cells become TE, inner PE Conservative division: Always only 1 TE into 2 TE First differentiation ICM retains pluripotency, goes to inside. TE develops on outside Contributing factor: position of blastomeres in zygote (inner become ICM more likely, outer become TE more likely) and polarity of transcription factor in cells Blastomeres become polarized along apical-basal axis before TE:ICM division Cells divide into outside (TE) and inside (ICM) cells according to polarization Pluripotency genes (Oct4, Sox2, Nanog) downregulated by Cdx2 in outside -> Absence of these make outside cells TE

Inside outside asym.

Cells can sense if they are on outside or inside via hippo signaling cascade, via degree of contact to other cells. Hippo signaling cascade: Cells sense if they are inside or outside. Tead4 complex is present in outside and enhances Cdx2 Cell polarity: Asymmetric division localizes Cdx2 on apical side of cell. More Cdx2 makes more TE. Decrease of Cdx2 through asymmetric division on inner side leads to Tead4 repression of Cdx2 and makes ICM. Cells retain pluripotency. Conclusion: Spacial orientation provides cells with level of CdX2 H3K27me3 enriched in ICM compared to TE. Help keep it differentiated. Methyltransferase responsible for methylating H3 leads to elevated Nanog and Sox2 -> ICM Greater on animal pole compared to vegetal, marks chromosome to be in active state, methylates H3 at Arginine 26. Needed to activate cell proliferation, by modifying histone -> Elevated levels of Carm1 make cells proliferate more. Carm1 overexpression leads to ICM/epiblast formation Levels of H3R26me Values are relative to each other: 1 AV cell at 1.0, 1 AV cellat 0.7, 1 A cell at 0.6, 1 V cell at 0.4

Histones Carm1

Blastomeres with highest CDx2 levels are daughters of cells with lowest chromatin modification. Asymmetric dimethylation of Arg17&26 on Histone 3 Blastomeres with Animal and vegetal parts have higher H3 modifications Blastomeres with only vegetal have lower H3 modifications -> also less pluripotent

Embryo proper

Made from ICM at 16 cell stage + outer cells of morulla during transition to 32 stage cell

64cell stage (blastocyst) ICM (13 cells) and trophoblasts in two separate layers (blastocyst) ICM forms into PE aand Epiblast, that rearrange SECOND DIFFERENTIATION: Formation of primitive endoderm and epiblast

ICM secrete protein Fgf4 to stimulate trophoblast cells to divide Second differentiation ICM in contact with blastocyst cavity becomes PE Deeper ICM keeps pluripotency and becomes epiblast ICM composed of cells with mixed epiblast and PE progenitors that segregate into layers PE differentiation needs Gata4,6 activation, antagonizes Nanog PE upregulated and loss of pluripotency Social mobility model Hatching 1st wave makes EPI, 2nd wave makes PE, then pattern Blastocyst digests hole in zona pellucida and squeezes through as if expands Uses trypsin like protease secreted by trophoblast Implantation First attachment blastocyst makes contact with uterus labile, mediated by L-selectin on trophoblast cells adhering to sulfated polysaccharides on uterus wall Polysaccharides synthesized in response to estrogen and progesterone secreted by corpus luteum Second attachment Stabile attachment, other adhesion systems come into play Trophoblasts make integrins -> bind to collagen, fibronectin, heparin sulfate, protoglycogen P-cadherins on trophoblast and uterus dock embryo Hut proteins make trophoblast secrete proteases to eat cell wall and bury itself Gastrulation Blastocyst becomes egg cylinder

Transcription factors by stage Mammals Non mammals Functional copies No germ plasma with material Germ cells determined by material in egg cytoplasm +/+ mater

Non-functional copies -/- mater

Maternal factors

Inherit maternal transcription factors, progressively degraded by RNA-induced silencing complex and replaced by zygotic transcription products. Take longer. Markers on H3 are depleted, leads to opening.

Paternal factors

Morula Zygotic transcription Egg-8 cell stage Morula stage Initiated early in mammals, at time when cells can switch fate. Go into full swing Each blastomere has CDX2 and Oct4 transcription factors Trophoblasts synthesize homeodomain containing, Cdx2 down regulates Oct4 and Nanog. Cdx2 activation regulated by Yap protein ICM synthesis Lats to phosphorylate/ap. Prevent CDx2 transcription Oct4 prevents Cdx2 transcription Cavitation TE secrete fluid into morula to create blastocoel. TE have sodium pump that pumps Na+ into cranial cavity. This draws in water osmotically -> enlarges. Pump stimulated by oviduct cells Blastocyst Blastocoel expansion pushing ICM to side of trophoblast cells -> blastula NOT FOUND IN NON MAMMALS Crucial in breaking pluripotency, becomes enriched at apical poles. Then it creates a positive feedback loop where cells with CDx2 are more likely to become TE. History (Av, A and V cells) influence Cdx2 levels Outside cells contain TEAD4 and enhance Cdx2. Together Tead4 and Cdx2 inhibit Oct4 and Sox2

CDx2

Cdx2 highest in daughter cells that show lowest levels of chromatin modifications, vegetal cells. Restrain pluripotent effect of cell. Regulating, activating gene for trophoectoderm and suppresses ICM expression Oct4 IN all early cleavage blastomere nuclei, but then only in ICM Important for survival of epiblast Nuclear localized. In general all transcription factors need to go to nuclear to transcribe. (b-catenin not always found nuclear, but goes in to transcribe) Primarily in ICM. But increased expression in PE cells. Level of Oct4 might make a difference in what cells become. Experiment to determine level Oct4 genes in 5 exons. Replace exon 2-5 with Dy IRES beta-geo (LacZ, NeoR) Ribosome translates transcript and make beta-galactosidase -> stains blue where gene is expressed. The amount of lacZ staining showed if Oct4 homozygous or heterozygous. Heterozygotes will express only 1x as much LacZ Homozygotes will express 2x as much LacZ Lack of oct4 is allowing Cdx2 to be more promoted. Oct 4 usually suppresses Cdx2, so if Oct4 is gone Cdx2 increases If you take ICM and take out Oct4 it becomes TE Level of Oct4 Lose a copy of Oct4 -> goes to TE Retain all of Oct4 Elevate or add Oct4 -> stay pluripotent -> tends to become PE cell

IRES sequence Nanog

Level of oct4 determines cell fate Allows ribosome to attach. Critical in maintaining pluripotency of stem cells. Begins expression in ICM, reverses epigenetic chromatin markers -> maintains pluripotency Found by: Transfect a cDNA expression library into ES cell Looked for colonies that stay pluripotent after removal (LIF suppressed) Found Nanog, which allowed ES-cells to remain stem cells even after Leukemia

inhibitory factor is removed ICM Histone marks enriched on ICM, Oct4, Sox2, Nanog retain pluripotency and resist differentiation TE Histone activated, make Cdx2 Cdx2 and Eomes upregulate to trophectoderm, loose pluripotency Cell contact If outer cells put inside no Yap and Cdx2 can form since there is more cell contact and signal pathway changes. (hippo gets activated). When cells divide, region in the middle suppress Cdx2 because more cell contact. Both Oct4 and Cdx2 is increased when blastomere is singled out and looses Yap localization Correct gene expression requires cell contact. Yap Factor database All these factors dont initiate specificity, they just support decision. Transcription factors reinforce cell fate by activating tissue specific gene expression and by promoting chromatin changes needed to maintain cell lineages. Name Cdx2 Oct4 Nanog Location 1st blast,then TE 1st blast,then ICM ICM Function Downregulate Oct4&Nanog Retain pluripotent ICM fate Prevents making hypoblast, makes epiblast. Self renewal in epiblast Regulated by Activation by Yap protein + Tead4 Downregulated by Cdx2 Downregulated by Cdx2 Precedes in nucleus before Cdx2 expressed. Overlaps with Cdx2

Stat3 Yap pr.

ICM ICM, TE Cofactor of Tead 4, enters nucleus of trophos to help Tead4 Maintains ICM integrity by promoting Oct4 and Nanog. Help establish ICM Acts upstream of CDX2 in TE Activated by Yap only in outer

SALL4

ICM

Tead 4

ICM, TE nuclei

development. Help establish ICM Lats pr. ICM Kinase, phosphorylates Yap protein and prevents it from going to nucleus

compartment when hippo is off Activated by cell surface kinase hippo

Fate specification Tead 4 No Tead -> mice died in development, die because implantation and swelling in uterus. Tead -/- No trophoectoderm, Oct4. Tead +/+ TE cells and Cdx2 and Oct4 Tead4 needed to go implant. Having one copy of Tead4 is enough to make it differentiate. Overexpression of Tead4: Stem cells become TE, activates Cdx2 in ES cells. Inner cells start to express Cdx2. In experiment they fused Tead4 to activating viral domain to constantly keep it on. Lats If knock out both lats 1 and 2-> cells become TE and all inner cells express Cdx2 Overexpress -> No TE only ICM Klf4 c-Myc MAMMILIAN HIPPO Makes Nanog In Cancer line retains pluripotency

DROSOPHILA HIPPO - Activation of hippo pathway limits cell proliferation Technology Twins & Chimeras Early cells of embryos can replace each other and compensate for a missing cell At 2 cell stage blastomeres capable of making two different individuals, but not at 4 cell stage. ICM cells have same pluripotency potency in all cells -> rise to same type of cell but not TE Fates of ICM determined by interaction among decedents Can inject ICM into blastocysts to create new embryo Chimeric mice aggregate 4-8 cell embryo can go into one How to: Take out zona pellucida with pronase. Put three embryos into a dish. Aggregate forms common blastocyst ICM cells can produce any cell in body. Ground state for stem cells ICM can just grow and not differentiate in certain conditions -> called ES, embryonic stem cells Embryonic stem cells: From Epiblast. Leukemia inhibitory factor (stat) or 2i (Fgf and GSk 3 inhibitor). Use both inhibitors: 1st inhibits G-coupled receptor and 2nd

Stem cells

inhibits Gsk3b (function to inactivates wnt signaling). Both needed to keep stem cells pluripotent. 2i activates wnt and maintains pluripotency. Extra embryonic endoderm stem cell: From PE cell. Use Gelatin and feeder medium Trophoblast stem cell: From TE. Use FGF4 and Heparin and feeder conditioned medium Stem cells require to grow on top of other stem cells. And grow in an environment that gives them feedback so they keep on expressing the right factors. Experiment: Put stem cell into blastocyst -> organizes into embryo Put into mouse back (organism) -> organizes into terratoma cyst Somatic nuclear transfer Briggs and King. Put nuclei from somatic cell of embryo and put into oocyte to see if nucleus ends up being reverted back to an earlier time period. Conclusion: If took nuclei from animal cap as donor nucleus -> high percentage to be able to develop into tadpole. If took cell from later stage -> low percentage able to become tadpole The later he came in tadpole development the lower the percentage of embryo developing normally. Suggests there is something irreversible happening. Preimplantation genetic diagnosis, test genetic disease before goes in uterus Chorionic villus sampling, sampling 8-10 weeks of gastation Take samples of placenta, analyze for genes Implantation tech. Screen embryonic cells before implanted into womb 2 blastomeres removed from embryo during 6-8 cell stage (no danger) FISH, fluorescent in situ hybridization determines whether normal number of chromosomes present PCR used to determine presence or absence of genes Sex selection Sperm selection Use PDG X chromosomes larger than Y

PGD Prenatal diagnosis

Separate via flow cytometer Chromatin immunoprecipitation embryo Allow examination of loci-specific histone modification in

Isolates complex that interact with gene RTPCR Western blot Looks at abundance of RNA. RNA reverse transcribed into DNA compliment Detect samples of proteins in given tissue Do a gel and then stain them. Gene trap insertion Inserting DNA chunk into gene Disrupts function, insert reporter gene (beta-gal) Anything downstream of insertion is lost -> mutant is non-functional Video imagining Tunel staining correlative evidence Detect fragmented chromosomes that are indicative of apoptosis. Less apoptosis, lss tunel staining provide 100000s embryo type tissues When Embryonic stem cells put into mouse ICM of blastocyst the ES makes embryo. Humans ES contributes to trophoblast in mouse ES does not In vitro Knock-outs Transgenic Genome sequence Inbred mouse strain Take cells prior to organogenesis to study development outside of organism Remove or replace a gene, look at effect Introduction of new gene into mouse, look at expression/over expression Look for gene, or related gene. Model knockout All individuals are genetically identical Good to make conclusion based on phenotype Often phenotype expressed in different levels in individual Use inbreds to eliminate genetic differences as factor Holding pipette sucks cell into places Drilling pipette sucks out nucleus

Stem cells Es

Nuclear transfer

Solter experiment

Then transfer fully differentiated somatic cell nucleus into empty hull Control Removed one male and put in another male chromosome. 18/348 survived. 100% blastocyst stage. Result: Huge tissue looks like placenta. Bi Paternal Remove female, add male nuclei. 0% survived, 53% blastocyst stage. Nonfunctional due to loss of maternal genomic imprints and maternal global genome demethylation -> leads abnormal gene expression to trophoblast. Paternal expression at twice the normal Hydatidiform mutation in nalp7 (similar to MATER) IFG2 and H19 expressed (normally only in maternal) in 2 paternal cells thats why the abnormal gene expression. Nalp7 unique to humans, mice have mater Bi Maternal remove male, add female nuclei. 0% survived, 73% blastocyst stage. Result: Small placenta, weird embryonic tissue. Conclusion: There has to be a difference in maternal and paternal genomes.

Tunnel DIAP1 DAPI stain

Stains apoptotic cells Inhibitor of apoptosis. Shows all DNA expressed. Immunostaining. Used to show congression failure of chromosomes in cells that were affected by BPA Was not convinced eggs could not do remodeling. Thought chromosomes maybe did not have enough time. Took frog foot webbing nuclei and put it into enucleated egg, grew it to blastocyst. But blastocyst nuclei into oocyte and so on. Serial transfer and then let it grow to tadpole Result: Somatic nucleus can be reprogrammed by oocyte cytoplasm USE experiment to make stem cells: Take nucleus out of egg and take cell from patient and put it into egg. Egg lets cell reprogram. Cell becomes blastocyst and then take out blastocyst ICM to get stem cells.

Gurdon experiment

Yamanaka experiment Made stem cells by adding gene. Generated induced pluripotent stem cells (iPS) He took cells from skin. Added reprogramming factors (oct4, sox2, klf4 and cMyc). Those cells became pluripotent stem cells. Then he put them in different media to grow different cells. How: Used viruses as vectors Assay ability to be pluripotent by making them grow into tumors to see what

type of cells they would make. Problem: Could mutate nearby gene by accident Solution: Now some people use drugs to activate factors into cells

Plasticity Aneuploidy 10-20% of human oocytes. Risk factors age, BPA, radiation, smoking, etc.

Aneuploidy observation Patricia Hunt, used immunofluorescence to staining DNA. Normal meiosis DNA aligned correctly in a metaphase plate (congression). In value cells meiosis DNA everywhere (M2 congression failure) Conclusion: Environment affected congression, in this case it was BPA in plastics. Disrupt reproductive physiology in female mice and sperm function Bisphenola, estrogenic compound analogous, used in plastic production Not fixed in plastic forever, leeches and gets exposed to organisms Longer the exposure higher the chance for chromosome anomalies Higher rates of tumors, breast tissue more sensitive to estrogen later in life

Nonyphenol BPA