BIO 325 Exam 1 Review

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Bio 325.1 Definitions Totipotenet- early blastomeres, up to 8 stage cell. Can for trophblast and embryo precurson cells Pluripotent- ICM, cant form trophoblast. Chorion- enables fetus to get oxygen and nutrients from mother. Secretes hormons and regulators of immunoresponce so mother doesnt reject it Epiblast- gives rise to all 3 layers SCMC MATERNAL GENE PRODUCTS, ASYMMETRICALLY LOCALIZED ON APICAL SIDE OF BLASTOMERE, MADEFROM FLOPED, TLE6, FILIA, MATER FISH determines wheather right number of chromosomes are present. Solter experiment Bipaternal remove female, add male nuclei. 0% survival. 50% blastocyst Hydatudufirn mutation in Nalp7 (MATER) Bimaternal- remove male, add female nuclei. 0% survival. 75% blastocyst. S Placenta
Lecture 7: overview of mouse Develepment Mammal embryo gastrulation is different than zenapus because the development is internal. Two pro-nuclei appear once fertilization take place in the zygote. Mouse cell division is slow. Week 4; all organ primordia (organ/tissue in earliest form) are present Fate of 20 human eggs o 20 eggs in contact with sperms o 17 successful fertilization o 14 successful implantation o 8.5 successful development to 4th week (gastrulation) o 6 fetus comes to term. (30% out of the 20) 10% of infant die in UK (civilized). 30% is due to birth defects CDC estimates that 1:33 affected by birth defect Genetic
Environmental Multifactorial- genetic composition and its environment Genetic mutations Multi gene or single gene effects o Syndrome- A mutation affects multiple organs o Pleiotropy- single gene affected o Mosaic pleiotropy Directly affects different organs. Gene A expressed in common precursor, present in both organs o Relational pleiotropy indirectly affect multiple organs through the action of one organ. Genetic heterogeneity- multiple genes affecting a pathway. Mutations in a number of genes can result in the same developmental abnormalities. Smad 4 acts on two different pathways. Abnormality of smad 4 would affect different pathways. Phenotypic Heterogeneity- variable phenotypes result from the same mutation. genes are not autonomous (alone), but rather are influenced by other gene products and environment. Abnormality of a gene might not affect phenotype. Rules of evidence: find it, lose it, move it Correlative evidence (find it) correlation between two events eg gene expression with differentiation. o observation occurring with development. Gene x correlates with a development, gene x leads to development. Weak evidence but leads to hypothesis Functional evidence (lose it) loss of function evidence. Does not exclude other possibilies Function evidence (move it) gain of function studies. Demonstrate that X causes Y. o Test to see what happens when you remove/gain structure Correlative and functional evidence must support each other to conclude. What makes the mouse a good model system? Life cycle- multiple generations in short period of time. Reverse genetics o Knockouts replace the gene and see effects o Transgenics introducing new gene under control of promoter. Effects of overexpression Forward genetics o Radiation o Transposons Tools o Genome sequence
Strains o Inbred- all individuals are genetically identical
Mouse life cycle 6-8 weeks o cleavage some cells differentiate some remain pluripotent o Implantation uterus. Pluripotent cells for epiblast epithelial layer, U shaped that will undergo gastrulation o Gastrulation o Organogenesis organ development and maturation Inactivation of the parental genome - gametes Gametogenesis process of gamete formation, differentiating Start marking their chromatin depending on if it is maternal or parental Sperm Imprinting- Sex specific marks o Modification of DNA DNA methylation happens when you have C next to G. ch3 on C. Sex specific Genome wide Histone deacetylation tail of histone becomes deacetylated. Open to close transitions. Transcription possible to transcription impeded state. Anatomy of unfertilized egg Zona pellucidia glycoprotein layer, acellular. o Surrounds until implantation o Function sperm recognition o Fertilization requires fusion of female egg, sperm o Egg has the female pro-nucleus (oocyte) and polar body o Polar body due to meiosis. result of asymmetric cell division. Fertilization and cleavage Egg has defense for multiple sperm entering Fertilization triggers completion of meiosis 2 o 2 pro-nuclei (nucleus of sperm or egg) o 2 polar bodies the axis will form where 2 pronuclei meet Left with Blastocyst o Has zona pellucidia o trophectoderm - Cell on outside implantation o Epiblast pluripotent cells make entire embryo o Primitive Endoderm. (hypoblast)- provides nutrients, absorb lipids Zona pellucida- prevents implantation in oviduct.
Demethylation of embryonic genome Genes have to re-activated (demethylation) Male actively become demethylated after fertilization Female genome (passive) after one division has 50% demethylation, requires a few divisions. Reactivation correlates with demethylation correlative evidence. Maternal protein is down 50% At first blastomeres are heavily methylated. The male is activaley demethylated, the female is demethylated through division so that most of the DNA is demethylated by the blastocyst stage. In zenapus, gene transcription beings at beginning of gastrulation. In mammals, it starts after fertilization. By 2 cell stage, the embryo is 100% transcriptionally active.
Lecture 8: Major findings: In the mammalian zygote, the maternal and paternal pronuclei do not contribute equally to embryonic development Aneuploidy (extra chromosomes) resulting form BPA exposure is attributed to congression failure (alignment of chromosomes at the Metaphase plate) during meiosis. Causes birth defects Maternal and Paternal pronuclei are not equal. Recap; mammalian preimplantation development DNA and histones modified to become transcriptionally inactive. CpG gets methylated, histones are deacetylated. Active; demethylated, acetylated deacetylation make chromatin compact and (methylation) DNA becomes silent, transcription cannot happen. Polar body marks animal pole, opposite is vegetal pole First division can occur perpendicular or parallel of AV pole If you take one blastomere from a 2 cell embryo, it will develop into a full organism.
If you take a blastomere from a 4 cell embryo, theres a big chance the fetus will not develop. 4 cell embryo, each blastomere have distinct potential. 8 cell stage- differentiation of blastomeres begin. Blastomere compact. You get outer cells that begin to look epithelial and inner cells that are not in contact with outside. Outside cells have apical microphilia. o Pumping fluid inside creating blastocyst cavity. Inner cells will become epiblast/ Epiblast is primitive epithelial layer. Epiblast will start gastrulation Primitive endoderm also epithelial absorption.
PIC 1
Derivation of tissues in human and mouse embryos
Inner cell mass blastocyst contribute to hypoblast layer and embryonic epiblast Epiblast will undergo gastrulation go into primitive streak to form 3 layers Trophpoblast will form placenta
Differences between female and male spermatogenesis Meiosis is started in female when its an embryo. Arrested in first meiotic prophase. Meiosis in the mouse oocyte (female) Leutenizing Hormone triggers release from meiosis rate Germinal vesicle (nucleus) breaks down Spindle migrates to peripehery important to make polar body. anaphase Subsequent cleavage releases a small polar body. Polar body divides into two. (metaphase II) Fertilization triggers the completion of Meiosis II
Genome equivalency: all cells in an organism are the same genome consisting for 2 copies of each chromosome (2n) Meiosis: production of cells with a single copy of each chromosome (1N) But. Are the paternal and maternal genemoes equivalent? Soulter used nuclear transfer. Use nuclear transfer to compare development with different parental origins. o 348 embryos replaced male pronucleus, 18 fetus survived. 100% to blastocyst o Bimaternal replacement is more successful than bipaternal replacement 75% vs 50% o Determined that the parental pronucleus are different Small placenta 2 maternal chromosomes Hydatidiform mole, primarly trophoblast (placenta) chromosomes primary derived from father - paternal o recurrent cases can result from mutation in Nalp7 (similar to MATER) o Nalp7 - maternal, deposited into egg. Something wrong with meiosis. Differences between methylation of maternal and paternal Methylation of CpG island observed imprinted genes- determines which alleles are expressed Maternal chromosome IGF2 is always off, paternal is on. o Mutated copy of paternal IGF2 would make organism not having IGF2 Region between genes ICR imprinting control region o A lot of CpG is present in ICR that are methylated on paternal chromosome o CTGF blocks these CpG on the maternal chromosomes in the ICR region to prevent methylation This isolates the gene (IGF2) from its enhancer, silencing the gene. Instead it activates H19 Put on during gametogenesis, this means that epigenetic marks (methylated) on imprinted genes are removed and replaced each generation. o No methylation Igf2 off. o Germ cells remove everything and start over with gametogenesis o Female will have demethylated chromsomes (gametogenesis) o Male germ cells with have methylated chromosomes (gametogenesis) Conclusion: maternal and paternal chromsomes are not identical.
Differential methylation of CpG islands observed near imprinted genes determines which allele will be expressed
Environmental risks of Aneuploidy Aneuploidy extra chromosomes Risk maternal age, irradiation, smoking + drinking, fertility drugs (high estrogen) 10-25% of fertilized human embryos have aneuploidy BPA contributes to aneuploidy rates Congression- normal alignment, chromosmomes aligned in middle Congression failure- the chromosomes are spread. o Location changed-different results. Lower congression rate failure. High levels of Estrogen can lead to congression failure. Bpa mimics estrogen, bpa leech out of plastic. o Bpa was what caused the rate of aneuploidy, higher congression failure. Hypothersized that endocrine changes during oocyte maturation could influence aneuploidy rates.
Lecture 9: Imprinting is set in gametogenesis and remains during embryogenesis. Only place where methylation is removed is in the primordial germ cells. Removal of methylation cpg in the 2-cell embryo o Maternal cpg demethylation through DNA replication. Gradual loss of cpgs methylation in genome o Thus, Embryonic genes have potential to become activated
At the two cell stage, blastomeres can contribute to inner cell mass and trophectroderm lineages. They are pluripotent. The later dividing blastomere contributes to more cells surrounding the blastocyst cavity By the four cell stage, the potential for individual blastomeres to sustain development of a mouse become restricted. o Isolated the cells to see if it would grow into a mouse. o Chimera Perpendicular animal and vegetal of Animal vegetal 85% could develop in animal Chimera of A cells- 25% Chimera of V cells 0% o Means: differences between the cells. 8-cell stage keeps its potecial of blastomeres to contribute to embryonic tissues. o Can get signals from neighbouring cells to instruct it. Despite restriction at 4 stage cell stage, the embryo can compensate for removal of one blastomere. o Genetic testing using: FISH PCR o Means the cells are not determined yet If a regulatory factor is asymmetrically distributed in a 2 cell blastomeresrotational cleavage can produce blastomeres with different cell fates o Mammal egg is very small compared to zyenapus. o Assumed that there were no maternal determinants. o If you had cell division occurring parallel or perpendicular to the AV axis, you would get different things. Concentration of Histone Arginine methyltransferase 1 (Carm1) is greater in animal pole cells compared to vegetal pole cells o Carm1 is an enzyme that transfers a methyl group onto arginine 26 on H3 histone. o Methyl group marks the chromosome to be in actively transcribed state. Epigenetic modifications at the 4 cell stage differ between animal and vegetal cells. o AV cells have higher levels of methylated (activated) histones compared to Vegetal o Hypothesizes that elevated level of histone H3 what makes the AV cells pluripotent forming epiblast Experiment: inject Carm1 + DsRed (fluorescent) into one blastomere of 2 cell stage o Observe what happens till blastocyst.
o Overexpression of Carm1 biases blastomere to become inner cell mass/epiblast. First differentiation we really see is compaction after 8 cell stage Compaction: earliest viable morpholical change in the embryo creates inner and outer cell Differentiative cell divisions establish the inner mass o microvilli on apical surface o Conservative division (parallel) one green two green o Differentiative division (perpendicular) one green one green one blue or red or purple o Differentiative division can create different cell types. Takes place at 16-32 cell morula. o Cells that keep the apical surface stay as trophectoderm cells Factors that are expressed in the apical surface and are found in the trophectoderm. Recurrent cases of hydatidiform mole, resemble trophectoderm. These cells have lost the maternal genome. Recurrent cases indicates mutation in Nalp7. Mater equivalent in mouse Mater is part of the . (SCMC) Does the subcortical Maternal Complex (SCMC) regulate cell fate division? Conservative or Parallel cleavage give rise to rophectoderm Differentiatve or Orthoganol cleavage give rise to trophectoderm and either epiblast or primitive endoderm. o Mater is found on the apical side of blastomeres. o Orthogonal cleavage will not form 2 blastomeres with the Mater. o Conservative Parallel cleavage will form 2 cells with Mater o Does Mater help define fate? SCMC priteins are maternal gene products that are asymmetrically localized with blastomeres. o Mater interacts with TLE6, Filia, FLOPED, make us SCMC. Maternal gene products (egg ) produced with growing oocyte. o Dictyate (resting) state maintained by follicle granulosa cells o Release from the follicle and ovulation driven by leutinizing hormone. In the ovary, FIGLA (factor in germline alpha) activates transcription factor of genes needed in the egg for development of the embryo. o Figlia will dimerize with Tcf3 to bind to enhancer o Attach to CANNTG o Target genes for egg development ZP1, ZP2, ZP3. o Missing this factor causes premature ovarian failure, early o Figlia transcribes genes that are needed for egg development. Use microarray to compare gene expression
o Looking for RNA only present in WT and lost in figlia mutant o Hypothesized that these RNA were important for controlling development o Identified FLOPED that was missing in the figlia mutants. What interacted with FLOPED. o Use of Co-immununoprecipitation to isolate a complex of proteins that interact with FLOPED. These proteins comprise the SCMC Fixed FLOPED and saw what attached Compare co-immunnoprecipitates from floped null and wild type ovaries o Found that in null, a lot of proteins did not attach. FILIA, MATER, and TLE6 did not attach This is now called Subcortical maternal complex o SCMC proteins accumulate in the egg and persists until the blastocyst stage- Maternally supplied mRNA. RNA of these proteins is found until the 1 cell stage. Porteins are found in blastocyst We dont know that they are forming a complex in the cell. o Gene trap insertion in the FLOPED gene disrupts the gene and incorporated a reporter gene (Beta-gal) Gene trap loss of function taking and inserting a chuck of DNA in middle of the gene to disrupt function. Anything downstream of gene trap will not get transcribed. o Maternal Floped null females are sterile despite normal everything else. Will have deplayed and asymmetrical cell division Abnormal cleavage, chromosomes not split evenly -/- are sterile +/- are not o FLOPED is localized in the egg cortex overlapping f-actin in the 1 cell stage after cleavage it is absent form the cell junctions. o FLOPED localization is dynamic and can redistribute if cell contact is lost. Cadherins are Ca dependent cell adhesion molecules If you move the cells in a low Ca environment, the cells split. The FLOPED localizes o FLOPED remained localized in the other layer through formation of the blastocyst. o FLOPED, MATER, TLE6, and FILIA co-localized in a SCMC on outer layer. Its a complex of proteins.
Lecture 10 ; cell signaling and activation of tissue specific transcription factors. hippo signaling suppresses expression of genes that encode activatros of trophoblast genes (Cdx2)
Lecture9- localized maternal factors- functional evidence that elevated levels of histone arginine methyl transferase favor inner cell/epiblast specification and correlative evidence that apical localization of SMCM may be involved in specification of trophectoderm. Ovulation triggers meiosis I How does cell position restrict cell fate? Cell position/interaction with other cells influence its fate. Basal/apical polarity activates cdx2, gene that is a homeodomain protein that is a transcription factor which activates gene needed for trophectoderm Decreased cell contact triggers hippo pathway in inner cell. Cdx2 is absent. o Hippo pathway on, suppresses cdx2 Tead4 is a transcription factor that activates Cdx2. Needs partner protein (YAP) Needed for embryogenesis. o Experiment: never had any -/- Tead4 in the embryos observed, meaning there was something wrong before. Hippo signaling pathway depended on cell-cell interaction Interacts to molecule that is fixed on another molecule Hippo pathway are conserved in vertebrates Activation of the Hippo Pathway limits cell proliferation Contact activated pathway FAT receptor\ Proteins EX and MER help pull complex to receptor HPO is a kinase, phosphorylates proteins, Works with Sav to hold proteins in complex This complex phosphorylates WTS which phosphorylates YKI YKI associatates to protein 14-3-3, it is ebiquinated and degraded. This means it will not go to the nucleus. Growth genes are not activated, slow proliferation rates. Inactive Hippo Pathway- cells proliferate and apoptosis is suppressed. SD is transcription factor. Can only bind DNA YKI is an activator of transcription, can only activate, not bind DNA. When SD and YKI bind together, result: activation of genes. Hippo pathway loss of function result in hyper proliferation. Tisuue overgrowth beings in the imaginal disk Tissue overgrowth in Hippo mutants result 2 things
1) Increase in cell proliferation and 2) Decreased apoptosis- cell death TUNEL = stains apoptotic cells small fragments of DNA DIAP1 = inhibitor of Apoptosis BrdU cell proliferation Increases in cell division also contributes to Tissue overgrowth in Hippo mutants Incorporation of T analogue DNA BrdU to detect cell division GFP wild type cell. Where there is BrdU means increases in cell proliferation Inactive Hippo pathway cell proliferation continues Apoptosis is suppressed Overexpression of Ex, Hpo, or Wts activates Hippo pathway reduces organ side. By activating apoptosis. Drice is important for apoptosis. Can be used to detect levels
Lecture 11: To become transcriptionally active, thhe DNA must be demethylated and the histones acetylated. The genome will then re-methylated genes that become silent. Loss of Hippo signaling results in tissue overgrowth. Transgenics experiment- overexpression Lats1 and 2 equivalent of WTS Yap equivalent of YKI TEAD4 equivalent of SD. active- Control of inner cells inactive control of outer cells TEAD4 knockout mutation caused there to be no implantation of the embryo trophoblast (implants embryo) did not develop. Asymmetry of the cells controls hippo pathway.
Cdx2 regulated all genes needed for trophoblast expression Hippo pathway activated in inner. Will phosphorylate LATS which phosphorylates YAP, will lead to break down. Activation of Cdx2 commits cell in trophoblastic linage.
Transcription factors characteristic of blastocyst lineages
Oct4 maintains pluripotent ICM Inner cell mass. Suppresses genes for trophoblast formation, Gata6 activates primitive endoderm genes Nanog activates epiblast gene. Over expression of tead4 triggers trophoblast gene expression in inner cells of morula. Inject Tead4 activator Tead4VP16 to activate the gene Yap found in the inner cell cytoplasm and the outer cell nucleus. Due to the Hippo pathway. Excluded from inner cell nucleus. Co-localized with Cdx2 in the outer cell nucleus. Should see Yap before seeing Cdx2 if it activates it. This is correlative evidence This is true when you stain it There are 2 lats. If you knock out one the other will work. Knocking them out will caused the whole cell to differentiate into trophoblast cells.
Overexpression of LATs, YaP is broken down, not found in the nucleus and Cdx2 is not activated.
Model of cell position-dependent fate specification in preimplantation embryos

In the polarized outer cells, hippo signaling is suppressed. [Yap] increases and together with Tead4 activates expression of trophoblast specific genes (Cdx2) Activation of hippo signaling inside triggers phosporylation and degradation of Yap. Tead4 without Yap cannot activate trophoblast genes.
Cell contact and cell polarity influence YAP localization Cell polarity (apical/basal) causes cell to express Cdx2. When you put two embryos together, the middle cells suppress cdx2 and YAP no longer exists in the nucleus.
o Need cell contact to keep epiblast, hippo pathway. Cell polarity is required for activation of cdx2 Separating a blastomere that had nuclear localized YAP, what you get is cytoplasmic YAP meaning cdx2 is not being expressed. Phosphorylated YAP.What is activated? Oct4 , nanong Separation cause increase in Oct4 expression, but a higher cdx2 level. o Confused cell o Putting it in a chimera, long separated cell can only differenciate into a trophoblast cell, short separated cell can differentiate in both.
Lecture 12 Nanog epiblast Gata6 primitive endoderm Oct4 keeps epiblast pluripotent but first it is found everywhere. Totipluroent can make any cell, including trophectoderm. Pluripotent cell is an ICM cell, can differentiate into all 3 germ layers. Cdx2 suppresses Oct4 and Sox2 that are needed to make the cell pluripotent. Becomes multipotent. Nanog suppresses Gata6
Stem cells from mouse blastocyst Embryonic stem cells must be kept from differentiating using: o Leukemia inhibitory factor (STAT) or o Culture with 2i (inhibitors) (fgf and Gsk3B inhibitors- inhibits beta catenin- wnt takes place) FGF inhibits g-coupled protein receptors GSK3B inhibits beta catenin phosphorylazes it. Inhibiting the inhibitor - Beta catenin is active and wnt is active. Activating wnt signaling will help preserve pluripotency. Experiment: Inject embryonic stem cells and label with lacZ into blastocyst. Will incorporate into epiblast. o Embryo will incorporate it. Mouse will carry the phenotype. Somatic cell nuclear transfer briggs and king test of genome equivancy concomitant with somatic cell differentiation there is a decrease in nuclear potenict. o Took a nucleus from various stages of development and implanted it into a empty oocyte. Wanted to see if it would reprogram. and develop into tadpole o Later he came into development, the lower the ability to support development. Pluripotent cells were good, multipotent cells (TE) were not
o Something irrevisable happening through development, cannot be reprogramed DNA methylation. Demethylation till blastocyst. After blastocyst, DNA methylation increases again into gastrula. John Gurdon o Serial Somatic cell nuclear transfer restores genome equivalency. o Kept sending the nucleus back to the egg and let it develop into the blastocyst. Did this over and over because he thought it just needed time. Then he let it develop into a tadpole. o Donor nuclei white frog. Transfer into egg of black frog. o By looking at progeny you could see it came from the donor nuclei. o Found: The somatic nucleus can be reprogrammed by the oocyte cytoplasm. o Reprogramming: undoing differentiation in nucleus
Induced pluripotent stem cells make pluripotent stem cells Reprograming factors Oct4, sox2, nanog. Maintain pluripotency Klf4 activates nanog.
Oct 4 is nuclear By the end Oct4 is more expressed in primitive endoderm o Differentiationg could have something to do with gradient of oct4 Oct 4 mutant embryos die around implantation Used IRES-Bgeo to insert after exon 1 for knockout. o IRES Will make lacZ enzyme and thus stain the emrbyos. o Look at level of oct4 and where Cross +/-, of embryos will not express oct4. Lack of oct4, levels of cdx2 go up and the embryo will turn into a ball of TE. Level of Oct4 expression in ES cells alters cell fate High Oct4 causes PE differentiation Medium causes pluripotent stem cell (epiblast) differentictiation Low level of oct4 causes TE differentiation Homeodomain- activates cascade of genes (cdx2)

BIO 325 Exam 1 Review

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nerdy-notes.

Strains o Inbred- all individuals are genetically identical

Derivation of tissues in human and mouse embryos

Transcription factors characteristic of blastocyst lineages

Model of cell position-dependent fate specification in preimplantation embryos

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