Developmental Biology Test 1

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Developmental Biology Test 1
2013-10-01 10:36:51

test 1
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  1. gametogenesis
    creation of gametes
  2. gamete
    haploid cell required for sexual reproduction
  3. haploid
    half the number of chromosomes, n
  4. diploid
    2n, 2 sets of chromosomes
  5. meiosis
    cell division process that divides cell from 2n to n
  6. fertilization
    merge 2 haploid cells to form a diploid zygote
  7. zygote
    one cell (first cell), diploid
  8. gastrulation
    • morphogenetic movement forming germ layers via signaling processes
    • begin to differentiate into 3 germ layers
    • cell movement
  9. 3 germ layers and what they become
    • ectoderm: outer, leads to skin and nervous system
    • mesoderm: middle, leads to circulatory, skeletal, muscular, lymph systems
    • endoderm: inner, leads to G.I. tract
  10. blastula
    • cleavage stage
    • embryo
    • ball of cells
  11. organogenesis
    formation of organs via germ layer interactions
  12. cleavage stages
    • little to no growth or change in volume
    • cell divides by mitosis to form a blastula
  13. how is an embryo organized?
    • cytoplasmic determinants: encode signaling molecules, establish bilateral symmetry, establish germ layers
    • determinants starts out in uniform concentration, then a slight perturbation elevates concentration in one position and they accumulate there
    • Establishing bilateral symmetry: 2 gradients with responses create 12 different regions
  14. germ cells vs somatic cells/tissues
    germ cell genetic information can be passed down
  15. how are germ cells determined?
    cytoplasmic determinant in the egg that programs cells that inherit it to become germ cells
  16. do germ cells start in the gonads?
    no, they have germ cell determinants called P granules and migrate to the gonads
  17. P granules
    • germ cell determinants
    • start out by being evenly distributed in the cell and then concentrate and segregate to one cell
  18. meiosis
    • modified type of cell cycle in which number of chromosomes is reduced by half
    • each chromosome replicated to form sister chromatids
    • first division event: cells have 4 chromatids
    • second division event: 2 chromatids separate to create haploid cells
  19. oogenesis
    • the process of formation of eggs
    • begins with an oogonia - pirmary oocyte - large primary oocyte - secondary oocyte and one polar body- ovum with 2 polar bodies
    • unequal divisions
  20. spermatogenesis
    • process that germ cells undergo if sex determination yields a male
    • begins with spermatogonia - primary spermatocyte - secondary spermatocyte - spermatids - spermatozoa
    • equal divisions
  21. yolk
    • granules of food reserve deposited in the oocyte and used for embryonic nutrition
    • amount determines how early cleavage will happen
  22. holoblastic
    • "holo" = complete
    • cleave completely into 2 cells
  23. meroblastic
    incomplete cleavage, so much dense yolk that cleavage furrows can't go through all of it
  24. cytoplasmic factors
    spindle formation
  25. animal pole
    • upper hemisphere of zygote
    • usually carries the polar bodies
  26. vegetal pole
    • lower hemisphere of zygote
    • rich in yolk
    • takes longer for cleavage furrow to go through this section so the cells made at this pole will often be larger
  27. cleavage
    • early cell divisions
    • no growth between divisions
  28. blastomeres
    products of cleavage
  29. maternal effects
    situation where the phenotype of the embryo corresponds to the genotype of the mother rather than its own genotype
  30. gastrulation
    formation of germ layers via cell movement
  31. types of cell movement
    • invagination
    • involution
    • ingression or delamination
    • epiboly
  32. axes
    • anterior-posterior
    • dorsal-ventral
    • left-right
  33. medial plane
    • separating left and right sides of body
    • center
  34. lateral
    away from center
  35. proximal
    • for appendages
    • near the body
  36. distal
    • for appendages
    • further away from the body
  37. morphogenesis
    creating of shape/form
  38. epithelium
    • sheet of cells on a basement membrane that acts as a barrier
    • specific interactions/specialized juntions between neighboring cells that keeps them close together
  39. mesenchyme
    • scattered cells
    • each cell is on its own and moves on its own
  40. cadherins
    • calcium-dependent: need calcium to adhere to each other
    • homophilic so want to bind to cadherins like themselves
  41. CAMs
    • calcium independent
    • homophilic
  42. Integrins
    • dont generally bind to other integrins
    • can interact with proteins on cells passing by, can act as markers
  43. microfilaments
    • polymers of actin
    • can be stress fibers, contractile ring in dividing cells
    • associated with motor proteins: myosin
  44. extracellular matrix molecules
    • glycosaminoglycans: constituents of proteoglycans
    • collagens
    • elastin: intermolecular cross-linking
    • fibronectin: bind to collagen and integrins
    • laminin: bind to collagen, etc
  45. condensation
    mesynchymal cells come together
  46. involution
    migration of cells around the edge of the constricted surface
  47. invagination
    cell sheet buckles so that the constricted region of cells forms a protrusion into the interior
  48. cavitation
    • filled space is hollowed out
    • can happen from cell rearrangement or apoptosis of cells in cavity
  49. EMT: epithelial-mesenchymal transition
    epithelium cells become mesenchymal cells
  50. MET: mesenchymal-epithelial transition
    mesenchymal cells become epithelial
  51. convergent extension
    • cell sheets change shape
    • individual cells intercalate in between each other
  52. epiboly
    sheet of cells expands to surround another population
  53. How is cell movement achieved?
    apical microfilament bundles: at tip of cells the bundles constrict causing those ends to become thinner causing a bend in sheet of cells
  54. developmental control genes
    • mostly transcription factors or signaling molecules
    • specific sets control different body parts
    • 2 mechanisms of operation: bistable switch and morphogen gradients
  55. bistable switch
    • 1. gene activated by regulator (needed to start but then not needed)
    • 2. gene activated by self (product) after regulator is gone
  56. morphogen gradients
    • morphogen: causes change in shape
    • morphogen is produced at specific source site
    • morphogen diffuses away from source creating a gradient
    • sink: site where morphongen is being destroyed
    • there are certain thresholds of amount of morphogen needed to activate certain genes
    • manipulation of gradients can alter forms
  57. Induction
    the means by which a gene product is either induced or inhibited
  58. competence
    the ability of a cell to take up extracellular DNA from its environment
  59. types of inductive interactions
    • paracrine: produce signal to induce changes in nearby cells
    • juxtacrine: signaling that requires close contact
    • autocrine: cell secretes a hormone or chemical messenger that binds to autocrine receptors on that same cell, leading to changes in cell
  60. notch/delta
    • juxtacrine signaling
    • notch is the receptor and delta is the ligand that binds to it.  when they bind, notch interacts with a protease causing a conformational change in notch.  this results in a cleavage enzymatic reaction of notch which can then go and bind to CSL and p300 to initiate transcription
  61. TGFBeta signaling pathway
    • paracrine
    • TGFbeta binds to a type II receptor which then recruits and phosphorylates a type I receptor.  this type I receptor can then phosphorylate smads so it can bind to the coSMAD and act as transcription factors
  62. FGF signaling
    • paracrine
    • FGF binds to receptor protein on cell surface (heparan sulfate).  then Ras becomes phosphorylated (GTP to GDP) so now it can go activate the Raf protein by causing it to associate with cell membrane.  Raf phosphorylates MEK which then goes and phosphorylates ERK.  ERK enters the nucleus and activates transcription factors by phosphorylation
  63. hedgehog pathway (Hh)
    • paracrine
    • constitutively active
    • hedgehog binds to ptc and this represses the activity of another membrane protein, smoothened (smo).  smo then represses the proteolytic cleavage of Gli TFs by removing C-terminal region turning them into repressors
  64. Wnt pathway
    • paracrine
    • wnt binds to frizzled (frz) which then activates disheveled (dsh).  Dsh represses gsk3.  when active though, gsk3 phosphorylates beta catenin.  if gsk3 is repressed, beta catenin remains unphosphorylated and binds with Tcf-1 and gets into the nucleus
  65. what is incorporated into pre-mRNA?
    • start codon
    • exons
    • introns
    • 5' and 3' untranslated regions
  66. what gets into mature mRNA?
    • start codon
    • exons
    • 5' and 3' untranslated region
  67. promoter
    • DNA sequence location wehre RNA polymerase binds
    • also initiation factors and basal transcription factors: general TFs bind promoter
  68. enhancers
    • repressors or activators
    • bind specific transcription factors
    • cell type specific TFs bind enhancers
    • modular
    • stabilize initiation complex
    • identified with reporter genes
  69. cell-specific transcription factors
    • must have 3 functional domains: DNA binding,, protein-protein interaction, trans-activation (other proteins)
    • categorized by DNA-binding domain
    • activate or repress transcription
  70. homeotic
    transformation of one body part to another
  71. homeobox
    • DNA sequence that encodes ~120 nuceotides
    • conserved
  72. homeodomain
    • protein encoded by homeobox
    • ~60 amino acids
  73. hox genes
    • subset of homeobox genes
    • specify A-P patterning
  74. mutation
    change in DNA sequence
  75. loss-of-function mutation
    • mutant less active than WT
    • null: no function, no protein
    • allelic series: different levels of loss of functions, mutations are in different areas so different levels of function
    • usually recessive
    • can be dominant = haploinsufficiency
  76. gain of function mutation
    • mutant more active than WT
    • usually dominant
    • can be recessive: sickle cell
    • can be constitutive
  77. dominant negative
    • mutant protein not functional and interferes wiht WT protein
    • one mutant protein in a complex of WT proteins will inactivate whole complex
  78. maternal effect and zygotic mutations
    • maternal effect: depends on genotype of mother
    • -zygote doesn't have the required determinant
    • zygotic: depends on genotype of embryo
  79. temperature sensitive mutation
    • display mutant phenotype at nonpermissive temp (too high usually)
    • appear WT at permissive temp
    • often weak loss of function because proteins denature at high temperatures
  80. genetic mosaic
    • mixture of cells that don't all have the same genotype
    • gynandromorph
  81. screening for mutants
    • mutagenize male crossed with WT female
    • % of F1 will inherit mutant allele*
    • F1* crossed with WT
    • % of F2 will carry mutant allele
    • mate F2 siblings and look for crosses that give ~25% mutant phenotype in F3
  82. autonomous
    • affects self or cell that it is produced in
    • only affect cell where gene is expressed
  83. spatial control experiment
    • GAL4-UAS system
    • GAL4 is a transcription factor that binds to UAS and activates gene of interest
  84. mRNA injection
    • transcribe mRNA from cloned gene in vector in vitro
    • inject mRNA to early embryo (1-cell preferably)
    • assay embryos for developmental disturbances
  85. loss of function experiments
    • targeted mutagenesis via nucleases
    • zinc finger nuclease
  86. antisense morpholinos
    • morpholinos: synthetic construct that can be designed
    • inject at early stage of development, binds to mRNA before start site so blocks translation so protein will not be made: can see what happens to embryo when protein is absent
  87. RNA interference
    • RNAi
    • way to knock down wild type function
  88. targeted mutagenesis via nucleases
    • zinc finger nuclease: zinc fingers recognize triplets
    • -fokl nuclease forms dimer and cuts between 2 distinct ZF target sites
    • A TAL effector nuclease: similar to ZF but recognize individual nucleotides
  89. stage series
    • describe standard stages of development at a certain temperature
    • establish time between each stage and specific structures to look for
  90. fate map
    label cells or regions in early stages, follow to later stage to assess fate
  91. manipulating development
    purpose: see what differentiation factors affecting growth and development
  92. stages of commitment
    • specified: received signals, has gotten some indication of what developmental path it should take.  in isolation will adopt fate
    • determined: received instructions/signals, will adopt fate in isolation or if transplanted to a new location
  93. grafting and isolation experiments
    • donor tissue put in exact same position in another host (orthotopic graft)
    • -tells us cells are competent but don't know if they are determined or specified
    • heterotropic graft: donor cells put in different position on host
  94. Transplantation experiments
    cells have inducing molecules that can induce cells around them
  95. combination experiments
    there are determinants at vegetal pole
  96. instructive induction
    • signal subdivides tissue
    • -appositional: divides into 2 types of tissue
    • -morphogen: gradient divides into multiple types of tissue
  97. permissive induction
    signal changes cell fate but no subdivision of tissue
  98. identifying factors defining developmental commitment
    • expression
    • activity: where it's expressed
    • inhibition
  99. range of dissecting microscope
    10x to 50x
  100. range of compound light microscope
    40x to 2000x
  101. microinjection
    used to introduce: reporter genes, morpholinos, RNAi, transgenes, mRNA
  102. DIC (differential interference contrast)
    • allows for enhanced contrast in transparent specimen
    • requires polarizer and specialized prisms to manipulate light
  103. fluorescence
    • allows for visualization of fluorescence substance within a spectra
    • requires fluorochrome, dichroic mirror, filters
  104. confocal microscopy
    • allows for imaging of thicker specimens or whole mounts
    • requires laser to illuminate one place at a time so you don't get so much interference from out of focus fluorescence
  105. histology
    • preserving a specimen so it can be examined or manipulated in the future
    • -treat with fixative: make cells harder
    • -dehydrate: remove water by alcohol washes
    • -equilibrate in substance that allows manipulation
  106. microarray
    • to study gene expression
    • start with 2 different cell populations- extract mRNA
    • isolate cDNA and label wit fluorescent dye
    • hybridize to array
    • scan for fluorescence
    • indication of relative levels of cDNA
    • -green: untreated cells
    • -yellow: both
  107. stage series for mRNA or protein
    • extract mRNA at differnt developmental stages
    • label mRNA of interest and run on a gel
  108. RT-PCR
    • detecting specific mRNA
    • use reverse transcriptase, cDNA
  109. RNA-Seq
    • modern sequencing has become very efficient and much cheaper
    • sequence all cDNAs in a sample
    • compare cDNAs to genomic sequence
    • reveals frequency and type of cDNA in sample
  110. in situ hybridization
    • goal is to find an mRNA molecule
    • make antisense probe
    • DIG: digoxygenin, plant sterol, can be detected in mammals by antibodies
    • AP: alkaline phosphatase, antibody, become purple dye when phosphate is removed
  111. immunostaining
    • have an antigen we want (protein or carbohydrate) so create an antibody for it by injecting the antigen in an animal and letting its immune system produce the anibody
    • a second antibody is produced by another animal to detect the first
    • usually visualized by fluorochrome
  112. reporter genes
    • principle of detection
    • -have an enhancer, promoter, and reporter gene that can be visualized fused to gene of interest
    • microinjection of vector into embryo
    • why isnt reporter expressed in all cells? because reporter genes have an enhancer
  113. cell-labeling
    • cell membranes with vital dyes, Dil or DiO
    • cytoplasmic molecules with fluorescent dextran, enzyme-substrate
    • used for fate mapping, clonal analysis, other lineage experiments
  114. the big six model organisms
    • C. elegans
    • drosophila
    • zebrafish
    • xenopus
    • chick
    • mouse