DB Exam 1 Review PDF

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Developmental Bio exam 1 review Cell life cycle ‣ ‣

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during cleavage, zygote cytoplasm divides into many smaller cells called blastomeres; blastomeres form a sphere known as a blastula Gastrulation- extensive cell rearrangements that results in three germ layers: endoderm, ectoderm, & mesoderm ‣ after germ layer formation, cells rearrange to form organs; called organogenesis ‣ some organisms hatch/are born not sexually mature- must undergo metamorphosis ‣ the separation of somatic cells and germs cell is one of the first differentiations to occur in animal development ‣ Gastrulation begins at a point on the embryo surface that is roughly 180º opposite the point of sperm entry - formation of a dimple called the blastopore (future dorsal side) Blastopore expands to form a ring, cells migrating through the blastopore into the interior become the mesoderm and endoderm; cells remaining outside become the ectoderm Organogenesis in a frog begins when the cells of the most dorsal region condense to form the notochord; notochord cells produce chemical signals that redirect the fate of the ectodermal cells above it- instructed to form cells of the nervous system. during this stage, the embryo is called a neurula- later will form neural tube ‣ Mesodermal tissue adjacent to the neural tube & notochord becomes segmented into somites- precursors to back muscles, spinal vertebrae, and dermis ‣ holoblastic pattern of cleavage where egg is divided into successively smaller cells (frogs & mammals) ‣ meroblastic pattern of cleavage in chicks where only part of the egg is destined to become the embryo The pattern of embryonic cleavage peculiar to a species is determined by two major parameters: 1) the amount of distribution of yolk protein within the cytoplasm, which determines where cleavage can occur and relative sizes of the blastomeres; and 2) factors in the egg cytoplasm that influence the angle of the mitotic spindle and the timing of its formation ‣ yolk rich pole- vegetal pole ‣ yolk concentration in the animal pole is relatively low- cellular divisions occur at a faster rate here; zygote nucleus is often displaced toward the animal pole mesenchyme: connective tissue in an embryo retinoic acid- converts anterior limb bud tissue

DB Techniques Cloning by somatic cell nuclear transfer Oocyte donor- Enucleated egg (remove meiotic spindle) Nuclear donor- udder cells used, placed in culture (grown in G1 stage)

Transfer udder cell to enucleated egg & fused with electric current, culture for 7 days until blastocyst is formed, transferred to surrogate Immunohistochemistry; indirect and immunofluorescence indirect immunohistochemistry involves primary & secondary antibody, DAB & HRPO; detects protein over time immunofluorescence involves a florescent tag and primary antibody;antibody; transcribed and translated

In situ hybridization involves a complimentary strand of DNA, RNA or nucleic acids to localize a specific DNA or RNA sequence in a portion of tissue Reporter genes Microarray used for cancer biology, compares the expression of genes in one sample vs another; gene expressions that change over time RNA-Seq transcriptome: all mRNA at a specific point in time in a sample - this varies, using bioinformatics, you can see everything at once Transgenesis -changing the genome, put something in that isn’t normally there OR remove -bacteria plasmid used to deliver components Knockouts (reverse genetics) Select a gene> generate null mutant > phenotype RNAi RNA Interference - neutralizing mRNA molecules

Ways to regulate gene expression • Chromatin modification: methylation & acetylation • methylated tails establish condensed configuration- repress transcription • acetylated tails establish uncondensed nucleosome structure- promotes access and active transcriptions • Transcription regulation • transcription factor complex: changes shape of DNA, DNA region of enhancer & promoter change shape and fold into each other • certain transcription factors bind to DNA on the promoter whereas others will bind on the enhancer • transcription factors: proteins that bind DNA with precise sequence recognition for specific promoters, enhancers, and silencers • regulatory sequences are usually on the same chromosome as the gene they are influencing; cis-regulatory regions/ proteins, like dimmable switches • promoters: sites where RNA polymerase II binds to DNA sequences to initiate transcription • basal transcription factors: specifically bind to the CpG-rich sites and form a saddle that can recruit RNA polymerase II and position appropriately for the polymerase to begin transcription

• enhancers: DNA sequences that signal where and when a promoter can be used and how much gene product to make • silencers: prevent promoter use and inhibit gene transcription • High CpG-content promotors • developmental regulators • control transcriptional factor expression • typically ‘on’ during developmental events • turned off by histone methylation (Lysine) • Low CpG- content promoters • Active in mature cells • Carry out function/maintenance of cells • Typically “off” by DNA methylation (Cytosine) • Turned on by transcription factors • Major transcription factor families & subfamilies • Hox- axis formation • POU- pituitary development, neural fate • Lim- head development • Pax- neural specification, eye development • Master Regulatory Genes • Encode transcription factors • Regulate hierarchy of genes • determine cell fate • EX: Pax6 in flies • Yamanaka factors - can give rise to progeny of all three germ layers • Differential mRNA splicing: • Differential mRNA translation • Post-translational control Cell interactions Juxacrine interactions: (receptor & ligand are both membrane bound) • gap junctions • tight junctions • adherens junctions Paracrine signaling • close, local interactions • ligand is secreted • receptor is membrane bound Endocrine (classical hormone) • Travels long distance thru the blood stream • extracellular *cadherin linkage to cytoskeleton- importance of the amount of cadherin for correct morphogenesis, aggregate surface tension correlates to # of cadherin molecules in cell membrane. the type of cadherin expressed can result in different sorting behaviors Cell matrix interactions - PG

- FN: fibronecytin, membrane spanning receptor proteins that bind fibronextin on the outside of cell while binding cytoskeleton proteins on the inside of the cell - Laminin - Collagen Epithelial-mesenchymal transition (EMT): seen in vertebrate embryos during normal formation of neural crest from the dorsal region of the neural tube & during formation of the mesoderm Every signal transduction pathway has: - signal/ligand - receptor - effector

Important cell signaling pathways in development • RTK- activated by fibroblast growth factor, the receptor tyrosine kinase is dimerized by the ligand • Wnt • Hedgehog • Notch • Jak-stat- casein gene activation- secretion of hormone prolactin • BMP/TGF-b • Hippo

Central dogma & gene regulation Within the nucleus is DNA -> DNA provides instructions for protein DNA -> RNA -> processing -> exit nucleus mRNA undergoes process of translation -> amino acids -> folding -> additions to protein -> transported

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Gene structure - promoter - transcription initiation site - translation initiation site - leader sequence ( 5’ UTR) - introns and exons - translation terminator codon - 3’ UTR - Poly A addition side mRNA processing and translation - 5’ CAPS - 3’ poly AAA — regulates mRNA activity - Start & stop codons — AUG (start) & UAA (stop) Cell signaling pathways; molecules involved, roles of pathways during development, be able to predict phenotypes if these pathways are inactive or hyper active Lab examples Ortholog, homolog, paralog SHH paper Ectoderm: precursor to epidermis, brain, and nerves Mesoderm: precursor to connective tissue, muscle, blood, heart, kidneys, gonads, & skeleton, notochord Endoderm: precursor to the lining of the gut and respiratory system Gene vs DNA DNA: sum of all genes Gene: segment of DNA that codes for something Chromosome vs Chromatin Chromosome: pieces of chromatin Chromatin: complex structure tightly packed Allele vs Loci Allele: each form of gene found at a specific location (loci) Loci: specific point at which a gene is found Nucleosome vs Histone

Nucleosome: chromatin wrapping histone Histone: basic proteins found in Chromatin Limb Development Proximal/distal patterning Posterior/anterior patterning Where do the cells come from? Skin epidermis- ectoderm dermis- mesoderm -somite -dermatome Muscle Mesoderm -paraxial mesoderm -somite -myotome Cartilage, Tendons & Ligaments Mesoderm -paraxial mesoderm -somite -sclerotome (cartilage) -syndetome (tendons & aments) Bones Mesoderm -Lateral plase mesoderm -somatic Neurons Ectoderm -Neural crest Blood vessels mesoderm -lateral plate mesoderm -splanchnic

Limb field specification Hox genes -Anterior/posterior axis positioning -initiation of bud outgrowth Forelimbs - Hox 4/5 -RA -Tbx5 -Wnt2b

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-FGF 10 -FGF 8 Hindlimbs -Hox 8/9 -Pitx1 -Tbx4 -Wnt8c -FGF 10 -FGF 8 AER- apical ectodermal ridge • necessary for distal limb formation • influences limb pattern • mesenchyme and epidermis must interact (FGF10 from the mesenchyme induces AER formation; AER expresses FGF8 which influence the mesenchyme) • positive feedback loop to maintain FGF 10/ FGF 8 Cell elongation -rate of cell replication : more cells in one area -differentiation : cell shape change, caused by signals which is caused by gene expression -cell migration : cells move into limb bud region from other places along the plank (sometimes comes from somites) EMT- Epithelial-to-mesenchymal transitions of the epithelial mesoderm Planar cell polarity (Wnt)- WNT is critical for determining axis formation adhesion is important for dorsal axal formation of the cell FGF and Wnt changes the expression pattern- Hox teen for patterning forelimb

ZPA- Zone of polarizing activity SHH Dorsal ventral patterning: Lmx1b- dependent by Wnt7a Wnt & BMP signalling Wnt7a induces dorsal cell fates of the limb bud through Lmx1b BMP signaling functions thru Engrailed-1 (En1) to regulate ventral limb patterning homolog: similar because of descent othrolog: same gene; speciation paralog: duplication & divergence: gene A —> Gene A’ (hox genes & cadherins)...