Lecture 31: Differential Gene ExpressionDifferential gene expression—individual cells take on different roles by expressing different genes- Example—skeletal muscle cells and nerve cells have their own proteins to relay signals*Different cells transcribe different genes because they contain different transcription factorsTranscription Factors—products of genes are produces as the embryo divides into multiple cells Two general strategies for initiating differential gene expression in embryos:1. Maternally derived transcription factors become localized in the egg/embryoa. Maternal gene products—RNA and protein contained within the egg were produced by the mother’s genome during the process of egg formationb. Localizing all cells have same nuclei and gene but only in one daughter cell does it inherit the RNA and synthesizes the protein and activates expression of that gene i. Need binding sites for the certain TF to activate expressionHow does it localize mRNA to region of cytoplasm?- mRNAs have specific localization signal base sequence in their 3’-UTR. This sequence becomes associated with a motor protein that uses ATP energy to walk across a polarized cytoskeleton, the motor protein drags the mRNA along with ito example—actin filament or microtubule and the motor protein, myosin2. Cells can communicate with one another using signals that activate transcription factorsa. Signal from outside cell can make one cell behave differently than another cell that was originally identical to itb. Requires a signal and a receptor:i. Receptors located in cells plasma membraneii. Ligands: signals that pass btn cells for specific receptor proteinsc. Signal transduction pathway—activated by a ligand molecule that has binded outside the cell causing a sequence of biochemical event inside the cell and activate specific target genes within cellSignal Transduction Pathway JAK-STATPlays a critical role in immune system1. Cytokine—ligand that binds2. Ligand binding causes 2 subunits of cytokine receptor to dimerize in cell membrane a. Dimerization brings cytoplasmic tails together which are each bound by the protein JAK—kinase b. Kinase: enzyme that transfers a phosphate from ATPsubstrate, have a high degree of specificity and only phosphorylate certain amino acids on particular proteins3. JAK cannot phosphorylate itself or the receptor its bounded to but when the cytokine receptor dimerizes each JAK protein can phosphorylate the other JAK and other receptor subunit and both sides get phosphorylated  receptor autophosphorylation 4. After phosphorylation the tails now serve as binding sites for the cytoplasmicprotein—STAT, JAK now phosphorylates STAT as well5. Once phosphorylated STAT functions as a transcription factor dimerizes and then transported into nucleus were STAT dimers bind to specific enhancers and activate transcription of the enhancer’s genes Achondroplasia caused by mutant allele of FGFR3, arms and legs fail to elongate- > 99% results from dominant alleles of this gene- > 97% of alleles involve the exact same missense mutation, G380R- missense mutation—one AA change effects how one of the autophsphorylation receptor functions - G380R mutation results in a constitutively active receptor protein—the receptor dimerizes and autophosphoryalates even when FGF is absent - Normally FGFR3 receives signals that tell a few cartilage cells to turn into bone, continuing for > decade o Cartilage cells with the mutant receptor all convert to bone at once, leaving no cartilage for future bone elongation *Mutation took a receptor that normally takes an extracellular signal and turns it into an intracellular signal and allowed intracellular signal to occur with out extracellular signalRegulatory regions of eukaryotic genes are often complex and must integrate the presence or absence of numerous transcription factors to determine rate of the geneSynergy: multiple signals, represented by multiple transcription factors, must be present simultaneously for transcription to occur - Example—sea squirt, mesP is only expressed in embryonic precursors of the heart, and requires two transcription factors, the only overlap is in the heart precursor cells, where they synergizeModularity of Regulation: multiple enhancer sequences can activate transcription independently in response to different TF’s- Example—PitX1 gene is essential for the development of four different organs, Pixt1 transcription is activates by a different tissue-specific enhancer in response to TFs unique to that organLecture 32: Genetic Pathways Axis Formation Life Cycle of Fruit fly:- Embryo develops in 1 day from eggshell worm-like larva- Larva grows through 3 instar phases then becomes a hard pupa shell- Inside the pupal case: metamorphosis to make the adult flyAnterposterior (AP): Anterior (Head)  Posterior (Foot)Dorsoventral (DV): Ventral (belly)  Dorsal (back) Heidelberg screen revealed that two different, non-overlapping sets of genes control the development of the AP and DV axes in the fly embryo AP Axis- First established by asymmetric localization of maternal gene products at anterior and posterior poles of the embryo- Bicoid gene—localized at the anterior end (During egg formation)- Oskar gene—localized at the posterior end ^^^^^o Pole cells inherit Oskar protein and becomes embryo’s germ line- At 2 hours  fly contains monolayer of somatic cells surrounding a yolk massand pole cells that segregate from the monolayer due to oskar protein*Cases where maternal gene products can localize and makes regions of the embryo different and sets up aspects of structures of embryos before embryo expresses any of it’s own genes Bicoid & Oskar Cells- Both are initially found at anterior end of egg, but as egg develops microtubules grow from anteriorposterior end - Adapter protein links bicoid mRNA to the (-) ends of microtubules and does not move (stays at anterior)- Oskar mRNA associates with motor proteins and is transported to the (+) ends of microtubules, relocates to the posterior end of egg - Conclusion: bicoid and oskar mRNAs have localization sequences in their 3’-UTRso To prove this: engineered a fusion gene with oskar protein coding sequence fused to the bicoid 3’-UTRo When injected fusion mRNA localized to the anterior pole of the egg where it induced a second set of pole cells to form DV axis- Established by an extracellular signal that acts