Developmental Biology Test 1

creation of gametes

haploid cell required for sexual reproduction

half the number of chromosomes, n

2n, 2 sets of chromosomes

cell division process that divides cell from 2n to n

merge 2 haploid cells to form a diploid zygote

one cell (first cell), diploid

• morphogenetic movement forming germ layers via signaling processes
• begin to differentiate into 3 germ layers
• cell movement

• ectoderm: outer, leads to skin and nervous system
• mesoderm: middle, leads to circulatory, skeletal, muscular, lymph systems
• endoderm: inner, leads to G.I. tract

• cleavage stage
• embryo
• ball of cells

formation of organs via germ layer interactions

• little to no growth or change in volume
• cell divides by mitosis to form a blastula

• 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

germ cell genetic information can be passed down

cytoplasmic determinant in the egg that programs cells that inherit it to become germ cells

no, they have germ cell determinants called P granules and migrate to the gonads

• germ cell determinants
• start out by being evenly distributed in the cell and then concentrate and segregate to one cell

• 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

• 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

• process that germ cells undergo if sex determination yields a male
• begins with spermatogonia - primary spermatocyte - secondary spermatocyte - spermatids - spermatozoa
• equal divisions

• granules of food reserve deposited in the oocyte and used for embryonic nutrition
• amount determines how early cleavage will happen

• "holo" = complete
• cleave completely into 2 cells

incomplete cleavage, so much dense yolk that cleavage furrows can't go through all of it

spindle formation

• upper hemisphere of zygote
• usually carries the polar bodies

• 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

• early cell divisions
• no growth between divisions

products of cleavage

situation where the phenotype of the embryo corresponds to the genotype of the mother rather than its own genotype

formation of germ layers via cell movement

• invagination
• involution
• ingression or delamination
• epiboly

• anterior-posterior
• dorsal-ventral
• left-right

• separating left and right sides of body
• center

away from center

• for appendages
• near the body

• for appendages
• further away from the body

creating of shape/form

• sheet of cells on a basement membrane that acts as a barrier
• specific interactions/specialized juntions between neighboring cells that keeps them close together

• scattered cells
• each cell is on its own and moves on its own

• calcium-dependent: need calcium to adhere to each other
• homophilic so want to bind to cadherins like themselves

• calcium independent
• homophilic

• dont generally bind to other integrins
• can interact with proteins on cells passing by, can act as markers

• polymers of actin
• can be stress fibers, contractile ring in dividing cells
• associated with motor proteins: myosin

• glycosaminoglycans: constituents of proteoglycans
• collagens
• elastin: intermolecular cross-linking
• fibronectin: bind to collagen and integrins
• laminin: bind to collagen, etc

mesynchymal cells come together

migration of cells around the edge of the constricted surface

cell sheet buckles so that the constricted region of cells forms a protrusion into the interior

• filled space is hollowed out
• can happen from cell rearrangement or apoptosis of cells in cavity

epithelium cells become mesenchymal cells

mesenchymal cells become epithelial

• cell sheets change shape
• individual cells intercalate in between each other

sheet of cells expands to surround another population

apical microfilament bundles: at tip of cells the bundles constrict causing those ends to become thinner causing a bend in sheet of cells

• mostly transcription factors or signaling molecules
• specific sets control different body parts
• 2 mechanisms of operation: bistable switch and morphogen gradients

• 1. gene activated by regulator (needed to start but then not needed)
• 2. gene activated by self (product) after regulator is gone

• 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

the means by which a gene product is either induced or inhibited

the ability of a cell to take up extracellular DNA from its environment

• 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

• 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

• 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

• 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

• 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

• 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

• start codon
• exons
• introns
• 5' and 3' untranslated regions

• start codon
• exons
• 5' and 3' untranslated region

• DNA sequence location wehre RNA polymerase binds
• also initiation factors and basal transcription factors: general TFs bind promoter

• repressors or activators
• bind specific transcription factors
• cell type specific TFs bind enhancers
• modular
• stabilize initiation complex
• identified with reporter genes

• must have 3 functional domains: DNA binding,, protein-protein interaction, trans-activation (other proteins)
• categorized by DNA-binding domain
• activate or repress transcription

transformation of one body part to another

• DNA sequence that encodes ~120 nuceotides
• conserved

• protein encoded by homeobox
• ~60 amino acids

• subset of homeobox genes
• specify A-P patterning

change in DNA sequence

• 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

• mutant more active than WT
• usually dominant
• can be recessive: sickle cell
• can be constitutive

• mutant protein not functional and interferes wiht WT protein
• one mutant protein in a complex of WT proteins will inactivate whole complex

• maternal effect: depends on genotype of mother
• -zygote doesn't have the required determinant
• zygotic: depends on genotype of embryo

• 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

• mixture of cells that don't all have the same genotype
• gynandromorph

• 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

• affects self or cell that it is produced in
• only affect cell where gene is expressed

• GAL4-UAS system
• GAL4 is a transcription factor that binds to UAS and activates gene of interest

• transcribe mRNA from cloned gene in vector in vitro
• inject mRNA to early embryo (1-cell preferably)
• assay embryos for developmental disturbances

• targeted mutagenesis via nucleases
• zinc finger nuclease

• 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

• RNAi
• way to knock down wild type function

• 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

• describe standard stages of development at a certain temperature
• establish time between each stage and specific structures to look for

label cells or regions in early stages, follow to later stage to assess fate

purpose: see what differentiation factors affecting growth and development

• 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

• 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

cells have inducing molecules that can induce cells around them

there are determinants at vegetal pole

• signal subdivides tissue
• -appositional: divides into 2 types of tissue
• -morphogen: gradient divides into multiple types of tissue

signal changes cell fate but no subdivision of tissue

• expression
• activity: where it's expressed
• inhibition

10x to 50x

40x to 2000x

used to introduce: reporter genes, morpholinos, RNAi, transgenes, mRNA

• allows for enhanced contrast in transparent specimen
• requires polarizer and specialized prisms to manipulate light

• allows for visualization of fluorescence substance within a spectra
• requires fluorochrome, dichroic mirror, filters

• 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

• 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

• 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

• extract mRNA at differnt developmental stages
• label mRNA of interest and run on a gel

• detecting specific mRNA
• use reverse transcriptase, cDNA

• 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

• 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

• 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

• 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

• cell membranes with vital dyes, Dil or DiO
• cytoplasmic molecules with fluorescent dextran, enzyme-substrate
• used for fate mapping, clonal analysis, other lineage experiments

• C. elegans
• drosophila
• zebrafish
• xenopus
• chick
• mouse