PDBio 382 Developmental Biology, Suli Winter 2021, Midterm 1

Describe how cloning shows that each cell has the same genome (genetic information, genomic equivalence).
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Describe how cloning shows that each cell has the same genome (genetic information, genomic equivalence).
Dolly sheep experiment
-You take an enucleated egg and transfer an udder cell grown in G1 stage into the egg.
-The egg and cell are fused with an electric current, and the embryo is cultured for seven days and a freaking blastocyst forms! The embryo is put back into the surrogate mother, and Dolly is born - who is genetically identical to the udder cell donor. Wild.

You got a whole sheep out of a single udder cell - not just another udder cell. So that showed that all any cell has the entire genome, so if you have the nucleus, you have access to all cell types.

All cell types in our bodies share the same genetic material.

This is called genomic equivalence.
Image: Describe how cloning shows that each cell has the same genome (genetic information, genomic equivalence).
How can gene expression be regulated at the transcriptional level?
This is how we get each cell to express different sets of genes; this is the differentiation of cells.

Essential components of a gene are:
-a promoter region
-transcription initiation/termination sites
-exons and introns
-untranslated regions

Basic requirements for transcription:
-Basal transcription factors:
-Assembled at promoter, recruit RNA Pol II, free nucleotides, and these are essentially the same for every gene in every cell.
What would happen if transcription in every cells depended on basal (the same) minimal transcription factors?
Teratomas would form - there is nothing regulating the activity.

We need something to make sure that there are regulation factors that regulate expression in specific cells of specific genes. These are ENHANCERS (which bind TFs that recruit nucleosome modifying enzymes)

You also can regulate differential gene expression with:
-Transcription regulation
-mRNA regulation (localization splicing, degradation, etc)
-translation
-post-translation modification
-protein degradation
What are enhancers and where can they be found in a gene?
Enhancers are regulatory DNA sequences that bind transcription factors (TFs recruit nucleosome-modifying enzymes, loop DNA, bring enzymes close to the promoter, and help start transcription).

They're found right before the promoter and also downstream of the promoter (in an intron).

Enhancers can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or thousands of nucleotides away.

These elements function at a distance by forming chromatin loops to bring the enhancer and target gene into proximity.
Image: What are enhancers and where can they be found in a gene?
How can enhancers restrict gene expression in specific tissues?
Tissue specific enhancers restrict gene expression in certain tissues.

In the brain, there are certain TFs that are expressed only in the brain - the brain-specific enhancer is recognized there.

In a limb, it's different. The LIMB-expressed TFs are binding to the limb enhancer in the same gene, but the TFs are only binding to the limb-specific enhancer.

The same gene is used in the brain and in the limb, but it is turned on by different TFs.
How can one use enhancers to drive exogenous gene expression (genes not found in that particular animal or tissue?)
Regulatory regions can be used to drive exogenous gene expression in specific cells.

Exogenous = a gene not expressed in that specific tissue or cell (like GFP or a neuronal gene in a fibroblast).

You can use an enhancer and a minimal promoter of a specific gene and its TFs to promote that's gene's expression in a different place.

Even though every cell has that enhancer, the only place that gene will be expressed is where those TFs for that enhancer are (like in photoreceptor cells). So you'll only see that gene expression in those photoreceptor cells.
What chromatin modifications can regulate gene expression?
Acetylation and methylation.
What are silencers and insulators, and how can they regulate gene expression?
Silencers are DNA regulatory elements that inhibit transcription.

Silencers stop specific gene expression.

Like NRSE (neural restrictive silencer elements) found in many neuronal specific genes in mice.

NRSF (neural restrictive silencer factors) bind to the silencer and stop that neuronal gene from being expressed in other cells. NRSF is found in non-neuronal cells!

Insulators are also stretches of DNA that also inhibit transcription; they set the boundaries of gene expression. They limit the range in which an enhancer can activate gene expression by changing chromatin structure.
How does alternative splicing regulate gene expression?

Can you give an example?
This is an example of post-transcriptional regulation.

Alternative splicing regulates gene expression by altering the order in which exons are spliced together to direct the synthesis of different protein variants (isoforms).

You can get many proteins from a limited number of genes.

Ex of bad splicing:

DSCAM genes (down-syndome cell adhesion molecule) allow a dendrite to form correctly; they have 115 exons and 38,000 potential gene variations.

DSCAM-independent repulsion makes sure dendrites don't overcrowd or overlap, while DSCAM-dependent makes sure dendrites in the same neuron don't crowd.

A lack of repulsion between dendrites comes from non-matching DSCAM isoforms (incorrect splicing).
How can mRNA/protein localization regulate gene expression?

Can you give an example?
The concentration of mRNA can affect gene expression.

Different genes can have different affinities for certain TFs, so you can turn certain genes on at a given concentration (some at low, some at high) - as you're directing which genes are turning on and off, you can change the phase of the cell (part of endo or mesoderm).

Gradients tell a cell what to become.

Just look this up - its like hunchback/bicoid cells.
How does methylation regulate gene expression? Can you explain the role of methylation in tissue differentiation? What is imprinting and how can it affect gene expression?Methylation is tightening of the histone, which down regulates gene expression. Methylation patterns vary in cell types. Imprinting: For certain genes, we only inherit one working copy. The other copy is epigenetically silenced in the egg or sperm. Certain genes are only silenced in the egg, some are silenced in the sperm. Imprinting can happen in one sister chromatid, and a mutation can affect that same gene in the other gene inherited from the other parent, so that gene is just altogether missing in the organism (Angelman Syndrome [mom's copy defective/missing] and Prader-Willi Syndrome [dad's copy "]).How can we use the knowledge we have of gene regulation to mis-express or manipulate gene expression in a particular tissue and at a particular place? Why might we want to do that?You can activate gene expression in places that aren't usually expressed. We can grow different proteins that we want if we can manipulate this process, like in gene therapies.Can you explain the Gal4/UAS system (use diagrams)? This is Tissue Specific Gene Expression! Describe two reasons why it is used.We are trying to mis-express Pax6 from (in?) the imaginal discs. The question is whether Pax6 is sufficient to produce an eye on its own. Pax6 is a regulator that creates eyes. Mis-expression of Pax6 forms an entire eye. Gal4 has to bind to the UAS (upstream activating sequence), and thus we see the expression of downstream Pax6. The enhancer from imaginal discs in flies turns on Gal4. If you have Gal4, you'll be able to activate the UAS and thus express Pax6. So you cross a fly with an enhancer before Gal4 with a fly with a UAS (Gal4 binding site) of Pax6, so you can a get a generation of flies that mis-expresses Pax6. Gal4 is only expressed where its enhancer is expressed (in the imaginal discs). You can use UAS and Gal4 as triggers for other things too, like preventing toxicity or activating a gene you want. You just have to do the Enhancer/Gal4 x UAS/desired gene cross with flies.Why might we want to generate tissue specific knockouts?You can find out how a certain TF or protein affects development. Getting tissue-specific prevents global-knockout that can caused unintended and generalized developmental delays.Can you describe the Cre/lox system (use diagrams)? This is Tissue Specific Gene KNOCKOUT! Why might it be useful to study development?BThe Cre-lox technique Used to study the role of certain proteins in development/in certain pathways (like Wnt3 in the limbs). Cre recombinase is mediated by loxP sites. Cre-recombinase brings Lox-P introns together and excises the exon. Where the Cre-recombinase tissue specific enhancer is present, cre-recombinase will be expressed, which splices out Exon 2. LoxP sites can only be utilized when enhancers are placed before the Cre recombination site. Basically: Put loxP sites around the gene of interest, and make sure a tissue specific enhancer (TSE) is before the Cre Recombinase, and then you can knockout the gene of interest. The possession of an appropriate enhancer and using the Cre/lox system allows one to remove the function of a gene at a particular time and place, and prevents global knockout. Cre allowed for this type of control because it requires a TSE (tissue-specific enhancer)Can you describe the usage of the Rosa26 locus in studying development? Rosa26 is a locus in the mice genome that is always turned on and in all cells.Exogenous gene expression using Rosa26. Rosa26 is a gene locus that is always on in all cells. We can use Rosa26 with two loxP sites that are surrounding a STOP codon before our gene of interest. So we could use a tissue-specific enhancer and a Cre/Lox system to excise the STOP portion in order to allow a Gene of Interest to be expressed. Guaranteed transcription of the gene you want to be expressed.NEW LECTURE What techniques can you use to determine gene expression in a particular time and place?Cre/Lox: specific, local gene suppression Rosa26: guaranteed transcription of the thing you want Space and Time: Tissue Specific Gene Expression/Conditional Gene Expression and Knockout: Enhancers: Enhancers, Silencers, Gal4/UAS, Cre/Lox and Rosa26Can you describe the in situ hybridization technique? This is good for learning the place of a gene's activation and if it plays a role in development. Does it test for mRNA or protein expression?In situ hybridization (mRNA localization) Use a protease and detergent to expose tissues Hybridization with anti-sense digoxigenin probe -Complementary with mRNA under investigation -Digoxigenin label on Uridine residues Wash then hybridize with anti-digoxigenin antibody -conjugated with alkaline phosphatase Wash then color the reaction -incubate with a compound that turns purple when a phosphate is removed Wherever the mRNA is in the cell, purple dye will appear.Can you describe the immunohistochemistry (antibody staining) technique? Does it test for mRNA or protein expression?This is used for protein localization Looking at antibodies. Use detergent to expose tissues Primary antibody against protein in question - usually grown from a particular species (rabbit, mice, etc) Secondary antibody: recognizes that species-specific domain of that primary antibody; can either have alkaline phosphatase (purple) or GFP. So, wherever the protein is, we're going to see fluorescence.What are the pros and cons of in situ hybridization and immunohistochemistry techniques?Antibody staining (immunohistochemistry) gives information about protein localization, but it can be pretty unspecific and it's difficult to get primary antibodies. In situ would tell more about noncoding mRNA (not translated into a protein); you can just generate the primers easily - so it's a lot easier than making antibodies for specific proteins (easier than immunohistochemistry).What is reverse genetics?Begin with a gene that is known/characterized Disrupt gene or its downstream products to deduce gene functionWhat reverse genetics techniques can you use to determine if a gene is necessary?1. Gene targeting -homologous recombination -CRSPR-Cas9 2. Inhibiting protein function -Dominant Negative 3. Inhibiting translation -siRNA -MorpholinoCan you describe two techniques for gene targeting (use diagrams)? (think homologous recombination, CRSPR/Cas9)Homologous recombination (has been used to knockout genes): -cellular response to repairing double stranded breaks in chromosomes by using the sister chromatid as a template for repair -also happens during meiosis for genomic diversity -SO, you make a targeting vector with antibiotic resistance in the DNA, and the cell will use it as a template for recombination. So you hope the cell recombines and puts the antibiotic resistant gene - replacing a certain exon you're knocking out in the process - into the cell via recombination, then you select for it by growing the cells in an antibiotic medium. Only the cells that have recombined properly will survive. CRISPR/Cas9: -CRISPR is a prokaryotic immune system against viruses and plasmids -Guide RNA (binds to Cas9 nuclease) is made of tracrRNA and crRNA (homologous to 20 NT of gene sequence) -crRNA binds to DNA, and Cas9 clips the DNA and forms a ds break -the cell repairs the ds break via non-homologous end joining, resulting in deletions or insertions, which lead to a frameshift and hopefully termination of that target protein. -if NHEJ doesn't occur, you don't see a difference.How does the Cre/lox technique fit in reverse genetics?You can knockout genes to start to figure out how a gene affects development of an organism.Can you describe the Dominant negative technique (protein targeting) (use diagrams)?Overexpress a molecule (a dominant negative) incapable of functioning but able to out-compete its endogenous (healthy) counterpart. Examples: -receptors without intracellular domains to interrupt signaling -TFs without transactivating domains (ex: express a TF that doesn't have the B-catenin binding domain, so the B-cat can't bind to the TF and transcription can't happen)Can you describe siRNA and antisense morpholinos and how they inhibit protein translation or splicing?siRNA: small piece of dsRNA that can be used to interfere with the translation of proteins by binding to and promoting the degradation of mRNA. -dsRNA that matches the mRNA of the gene is injected -dsRNA is chopped up by Dicer RNase III and makes a protein complex -dsRNA/protein complex act enzymatically to cleave homologous mRNA Antisense morpholino: inhibit protein production via 2 different methods; targets mRNA, so it's not really a long term effect like affecting the DNA directly would Translation inhibiting MO: binds to the ATG site, resulting in inhibition of translation. Splice-blocking MO: inhibits splicing between exons, leading to incorrect protein being produced (truncated or nonfunctional protein).What are the pros and cons of each gene reverse genetics targeting technique?What is forward genetics?Begin with a phenotype to explore Find and characterize novel genes affecting the phenotype You mutate at random until you get the phenotype you're looking for. You don't know which gene causes your phenotype. Good: unbiased identification of new genes, produces knockouts/hypomorphic alleles Bad: takes forever and takes a lot of effortCan you describe how to conduct a forward genetics screen (use diagrams)?Go: ENU male crossed with a WT female G1 generation all has different mutations. m1/WT, m2/WT, m3/WT, m4/WT G2 comes from a same cross with two similar mutants: makes males m1/WT, females m1/WT, males WT/WT, females WT/WT G3: cross brothers and sisters from the same generation; pair-mate crossings (no longer random); 1/4 population is m/m (shows mutant phenotype), 1/2 m/WT, 1/4 WT/WT. 25% should show the phenotype you want by G3.What techniques can you use to determine if a gene is sufficient for a developmental process? (Is expression of the gene by itself enough to drive/allow the appropriate developmental event?)Pax6 is sufficient to induce eye in drosophila B-catenin is not sufficient to act in a cell that lacks its transcription factor. ----- Transgenics: enhancers to express a gene of interest Gal4/UAS: express a gene in a different places to see if its compatible with life mRNA injection: can be used to rescue a phenotype/induce a particular developmental process. -inject mRNA to see if it rescues (fixes) a phenotype cDNA electroporation: -used to misexpress a gene -DNA injected into fluid just outside the part of the embryo where misexpression is desired -small electrodes placed around the region of interest -a brief current opens up cell membranes and drives DNA into the cells -Egg is incubated and transient expression of the gene can be observedCan you describe transgenesis?Transgenics: enhancers to express a gene of interest; process of introducing an exogenous or modified gene (transgene) into an organism.Can you describe the Gal4/UAS system?See other cardCan you describe the Rosa26-CRE/Lox system?See other cardCan you describe mRNA injection?See other cardCan you describe cDNA electroporation?See other cardLECTURE 3 What are two strategies for creating cellular diversity? What is mosaic (autonomous) development? What is regulative (conditional) development?Autonomous: each cell is inheriting some determinant from the parent; cytoplasmic determinants are unequally distributed in the cytoplasm initially and cell division partials out those determinants; cell fate is predetermined by what it inherents. Conditional: fate determinants are not partitioned, first cells are all equal, a cell's fate is determined by communication with its neighboring cells; this is INDUCIBLE.Does cell-cell signaling fall into autonomous or regulative development?Conditional/regulative developmentKnow the definition of an inducer, induction, responder, signaling molecule, competence, competence factor, instructive signal, permissive signal, reciprocal signalInducer: cell tissue that sends the signal to another tissue Induction: When a cell changes the behavior/fate of another cell Responder: Cell/tissue that receives the signal from an inducer Signaling molecule: Secreted molecule from an inducer that is received by a responder Competence: the ability of a cell/tissue to respond to a signal (has a receptor for a certain signaling molecule) Competence factor: molecules that are required to transduce a signal in the responding cell (like a receptor) Instructive signal: signal that is required to induce a certain fate Permissive signal: a signal that provides the right environment to allow a cell/tissue to take on a fate that has already been determined (ie survival factor, ECM) Reciprocal signal: the responding cell/tissue signals to its INDUCER, talking back, switching their rolesIn the example of the optic cup signaling to the head ectoderm: 1) what can you say about the optic vesicle? 2) what can you say about the head ectoderm? 3) is the head ectoderm competent to receive signaling from the optic vesicle? 4) is the trunk ectoderm competent to receive signaling from the optic vesicle? 5) can the head ectoderm receive signaling from any tissue or cells?1) if it is removed or replaced with foreign tissue, the lens will not form; therefore, the optic vesicle is an inducer that induces the formation of the lens. 2) it is a responder; therefore, the optic head is competent (able) to receive that signal. 3) Yes 4) No, because it doesn't induce the lens (no freaking eyes appear on the trunk) 5) See 2 dummy, yes.In the experiment from Fujiwara et al, 1994 where pax6 was knocked out tissue specifically (using the cre/lox system) in the optic vesicle, surface ectoderm or both, what can you conclude about the role of pax6 in lens induction?If Pax6 is knocked out in the surface ectoderm, the lens does not form. If Pax6 is knocked out in the optic vesicle but not in the surface ectoderm, the lens still forms. You can conclude that Pax6 has to be present in the ectoderm in order for the lens to form. It allows the ectoderm to become competent to respond to optic vesicle signaling. See this slide to understand better.What is paracrine, juxtacrine, autocrine and endocrine signaling? Can you give examples of each?paracrine: signal is initiated by molecules secreted into the EC space and influence nearby cells or far away Ex: hedgehog, Wnt, TgfB/Bmp, Jak-Stat, RTK Juxtacrine: When the signal is initiated by molecules bound to the cell membrane of a neighboring cell Ex: Notch, ECM Autocrine: SELF-SIGNALING, the signal transduction cascade in a cell is initiated by molecules transcribed within the same cell. Endocrine: signaling molecules travel through the blood to exert their effects Ex: hormonesCan you diagram the signaling pathways of Hedgehog, (canonical) wnt, tgfB/Bmp, jak-stat, RTK, delta-notch to the detail shown in the uploaded figures (see book or lecture slides)See book or lecture slidesHow can the same signal have different responses in different cell types?You need the appropriate receptor and the appropriate transcription factors in order to induce the proper signal. Know the example of Wnt signaling in the thymus vs the gut. => The thymus has a thymus specific TF, where the gut has its own TF. The wnt gene is transcribed, but each cell type has its own TF to make this process happen.Can you describe how the presence of different surface receptors can affect cell responses?If a cell has the right surface receptor (if it is competent to receive a specific signal), it can cause a cellular response. If it is not competent, it cannot generate that response. A cell needs the appropriate type of receptor, as well as the appropriate TFs, to cause a cellular response.Can you describe how a gut cell and thymus cell turn on different expression profiles even though they both respond to wnt signaling?They have different TFs to activate their specific cell-type genes. They receive the same signal, but the tissue specific transcription factors determine which set of genes (thymus or gut) is expressed.In the example of the gastrula stage tissue transplantation between a frog and a newt: Why does the transplanted frog ectoderm respond to newt mesoderm? What is the newt mesoderm secreting?Tissues can be competent to receive signals across species, but they can only respond with their own genetic program. Newt mesoderm is releasing a signal, and frog ectoderm can receive it (and vice versa) The frog only has its own frog genes to work with, so you're making frog suckers as apposed to newt balancers (you can't make newt balancers in a frog because the frog doesn't have the genes for it!) The cells are competent to receive the signal, but they don't carry out the same function in newt vs frog cells.Why does the transplanted frog ectoderm form suckers instead of balancers?The frog only has its own DNA to work with - it doesn't have anything that encodes for newt balancers, only suckers.What is the role of cell-adhesion molecules in cell-cell communication?Cadherins: calcium dependent adhesion molecules; cells that can interlock/communicate this way have the same cell adhesion molecules.Can you give some examples of cell adhesion molecules and what type of adhesion they promote?E-cadherin: early in development expressed in embryonic cells, later in development restricted to epithelial cells N-cadherin: in developing nervous system P-cadherin: in the placenta R-cadherin: for retina formation protocadherins: (lack attachment to Catenin) found in different tissues Cells can self-segregate by distinguishing their same cell types from other types by recognizing like-cadherins (in other words, they'll clump with cells that express the same type of cadherin as them)What is the role of the extracellular matrix in cell-cell communication? Permissive or instructive? Why?It's an insoluble network of secreted macromolecules Fibronectin: provides matrix to help things move Heparan sulfate: present extracellular signals (FGF for example) It is a permissive signal: a signal which provides the right environment to allow a cell/tissue to take on a fate that has already been determined.In the example of retinoganglion cell transplants to determine the role of the dak gene, is the dak gene required in the retinoganglion cells or in the environment? Why? What kind of cell-cell communication does this represent: cell-cell, cell adhesion, cell-extracellular matrix? (pathfinding/migration)What is leading to the following of an incorrect pathway? The dak gene has been mutated in this experiment. In the mutant, they observed misguided axons. Then they put the mutated genes into a WT, and it looked normal. Then the put WT cells into a mutant, and observed a mess. So the dak gene is required in the environment - it helps with ECM formation.NEW LECTURE: C. Elegans What are key events during the cleavage stage in general development?Embryos in this stage are called blastomeres Divisions are synchronous Cytoplasmic divisions without increasing volume G1 and G2 phases are not present = no cell growth during cleavage; "biphasic" Maternal Cyclin B regulates cell cycle (regulates mitosis-promoting factor)What are key events during the mid-blastula transition?First zygotic genes are expressed (instead of relying on maternal RNA) Cells start independently regulating their cell cycle Synchronicity of cell divisions is lost G stages are initiated allowing growthWhat are key points about gastrulation? (formation of mesoderm, ectoderm, and endoderm)Reorganization of blastula through cell movement Three germ layers are formed Head (anterior) to tail (posterior) axis becomes apparent First cells to enter gastrulation cells usually contribute to anterior structures Caudal structures may continue far into later stages while anterior structures continue development. Different mechanisms of cell movements are noticed during gastrulation: -Invagination: infolding of a sheet (epithelium) of cells -Involution: inward movement of an expanding outer layer so that it spreads over the internal surface of the remaining external cells -Ingression: Migration of individual cells from the surface into the embryo's interior; individual cels become mesenchymal (separate from one another) and migrate independently. -Delamination: splitting of one cellular sheet into two or more less parallel sheets; while on a cellular basis it resembles ingression, the result is the formation of a new additional epithelial sheet of cells. -Epiboly: movement of epithelial sheets (ectodermal cells) spreading as a unit to enclose deeper layers of the embryo.Why use C.elegans to study development?For one, their embryos are transparent, so you can see development happening. Rapid embryogenesis (16 hours) Relatively few cells types (959 somatic cells) Hermaphroditic Entire cell lineage has been traced Relatively small genome fully sequenced Simple genetics It is easier to study developmental mechanisms in a simpler model.How is anterior posterior axis established in c.elegans? Sperm Par proteins (Posterior:1,2 Anterior:3,6) P-granules (what are they?)tIn the first cleavage, you get AB cells and P1 cells. AB cells become somatic, while P1 maintain posterior fate and also give rise to germ cells. AP (anterior/posterior) axis determined by sperm entry location. 1. Microtubules organized by male centriole initiate arrangement within cytoplasm. -Male pronucleus moves to closest end, marking posterior. -PAR proteins partitioned to specific AP locations (Par3,5 anterior end, PAR2,1 to the posterior) 2. PARs proteins localize P-granules (molecules that suppress somatic fate - so they help develop germ cells) to posterior end 3. P-granule: transitional regulators that ensure posterior (stem cell) identity and are partitioned at each division to the P# lineage. P-granules inhibits somatic transcription, but allows stem cell genes to be present. DV axis determined by the relative positions of the AB and the EMS cells respectively. Left side specified by the contact of the MS and the AB.al cellAre SKN-1, PIE-1, PAL-1 important in autonomous or regulative development in C. elegans? Why?Autonomous (independent of signaling). Depending on which combination you inherit, you will have a certain cell fate. **** see screenshot, was being terrorized by desktop goose. P1 cell lineage is autonomous: fates determined by the relationship of inherited cytosolic factors (see diagram); no outside signals. Posterior embryonic determinants: -SKN-1: maternal protein and begins to degrade before the 3rd cleavage -PIE-1 and PAL-1 present initially as maternal mRNA and alter produced by the embryo. Where they go/controlling their function: -SKN-1 and PIE-1 partitioned to the posterior blastomeres by PAR genes during the 1st cleavage. -PAL-1 mRNA is found in all early cells but its translation is inhibited in anterior cells.If given the diagram with SKN-1, PIE-1, PAL-1 can you tell the development of which cells depend on each of these factors?Look at the Model for P1 Lineage DeterminationDoes regulative development occur in C.elegans?Yes.If given the diagram of C.elegans embryogenesis in the 1-3 cleavage stages can you reason through the following scenarios? 1. If P2 cell is removed, EMS cell divides to produce two MS cells; What does this tell you about the E cell fate?The cells are communicating. E cell fate is induced by P2.2. If the AB.a and the AB.p cells are reversed the embryo still develops normally. What does this tell you about each of these cells?They fulfill similar roles/ are identical to each other.3. If the P2 cell is removed, the AB.p cell does not develop properly; what does this tell you about the AB.p cell fate?P2 signals to AB.p, which leads to its posterior fate.4. What makes the AB.a cell capable of becoming an AB.p if switched?Good question.5. Pharynx tissue is generated by AB.al and MS cells. Pharynx tissue development in the MS linage is SKN-1 dependent in the MS linage is SKN-1 dependentWithout the MS cell, the tissue cell doesn't become specified - regulative development (some sort of a signal) is important at this point. The MS cell becomes specified due to SKN-1 presence, MS cell goes back and talks to AB.al cell to become pharynx. Only part of the pharynx will form without Ab.al present.6. If the EMS cell is removed no pharynx cells form, but if the AB.al cell is removed, the MS cell still forms pharynx. Propose a model for why this is so.EMS cell induces AB.al to express pha-4, which results in pharyngeal precursors from the AB.al lineage. MS lineage depends on SKN-1 to generate pharyngeal precursors.NEW LECTURE: Drosophilia What is a drosophila syncytium blastoderm?All of the cleavage nuclei are contained in the same cytoplasm. No cell membranes exist other than that of the egg itself.Is there cytokinesis in a syncytium blastoderm? Is there karyokinesis?Early mitotic division in drosophila embryos occurs without cytokinesis. Yes there is karyokinesis because there are a bunch of nuclei that form within the same cytoplasm.Is there cell-cell signaling in a drosophila syncytium blastoderm?No, there is no regulative development because there aren't other cells to talk to! That doesn't happen until the gastrulation stage (the next step).What are egg polarity/maternal effect genes? Are they zygotic or maternal genes? Can you mention some of them?Maternal effect genes: the phenotype of the offspring is determined by the genotype of the mother. Gene activation is seen early in blastoderm (blastula) stage. There are the bicoid mRNA and Gurken mRNA from maternal genes. Egg polarity genes are maternal effect genes.How is anterior/posterior polarity initially established in the fertilized drosophila egg?A-P Polarity: -maternal nurse cells (outside of the embryo/oocyte) make gurken mRNA; so these are maternal genes -Gurken mRNA localizes by the nucleus and gets translated, then signals to the Torpedo (Gurken receptor) on the uncommitted polar follicle cells -Follicle cells then signal back to "posteriorise" the embryo -PAR-1 recruited posteriorly and establishes microtubules. -Some maternal mRNAs travel towards negative ends of microtubules (bicoid) -Other mRNAs travel towards positive ends (oskar, nanos) -These proteins will lead to a cascade of events that pattern the embryos.Can you explain the experiment that proved that maternal effect genes are required in the mother? (think torpedo mutants)Maternal effect genes are provided by the mother (required in the mother) for the development of the embryo. You take an embryo from both a WT mother and a mother deficient in the torpedo gene. You switch the pole cells of the embryos (the zygotic genome) and replant the embryos (now with different pole cells) back into the mothers. So, the WT mother had torpedo-deficient embryos and the torpedo-deficient female had the WT embryos. The WT mother with the torpedo-deficient oocyte still produced normal dorsal-ventral axis offspring, while the torpedo-deficient mother with the WT embryos produced mutant offspring. This shows that maternal effect genes are required IN the mother for the development of the embryo. Pole cells are the zygotic genome (the sperm or oocyte) - the follicle cells (the genes that are around) are the ones who carry the maternal effect.How can gradients lead to different gene expression in different nuclei in the syncytium blastoderm? Can you explain the Lewis-Wolpert theory of gradients?Egg polarity genes are deposited in the oocyte by the mother and form protein gradients. Different genes are activated by specific concentrations of bicoid protein. Bcd RNA is localized at the anterior of the egg and forms an A/P gradient after fertilization. Bicoid (Bcd) turns on segmentation genes such as hunchback (hb). Orthodenticle mRNA creation gets activated by high bicoid concentrations, while hunchback mRNA is activated at lower genes. Protein gradients are zygotic effect genes (turning on genes in the zygote). Lewis Wolpert's theory of gradients: cell fate is determined by the concentration of a substance that is diffused across the polarity axis.What kind of a protein is bicoid? What is its role in embryo development? How can you test it?Maternal Bicroid turns on segmentation genes such as hunchback (hb). When you increase the concentration of bicoid, you'd alter the hunchback expression domain.How does bicoid regulate hunchback? What would happen to hunchback if bicoid is overexpressed? How does this fit with the theory of gradients?Bicoid is essential for anterior structure formation When you increase the concentration of bicoid, you'd alter the hunchback expression domain. It proves that the concentration of one protein affects the translation of others.How do egg polarity genes turn on gap genes? Are gap genes maternal genes or zygotic genes?GAP genes (Krupple) are activated or repressed by egg polarity genes. Egg polarity genes initiate the genetic cascade that leads to the activation of segmentation genes and eventually homeotic genes. They are expressed in one or two broad domains along the anterior-posterior axis (about 3 segments)What would the larval phenotype look like if one of the gap genes is mutated?All squashedWhat are pair-rule genes? Can you give one example? What would the larval phenotype look like if one of the pair rule genes is mutated?PAIR-Rule Gene (even-skipped) Expression of this gene in seven stripes further sub-divides the embryo. Pair-rule genes have large transcription regulatory regions with different modules or enhancers. You're missing the even-skip segments in the mutant.We talked in class about the transcriptional regulation of even-skipped gene. How is eve's expression turned on in different stripes? What do the different modules refer to? (enhancers?....) How can you determine if a module is responsible for regulating eve's transcription in a particular stripe?Combination of gap genes determines the activation of eve in a specific stripe. Different modules/enhancers are responsible for expression of eve in different stripes. You can hook up a module to a reporter gene (like LacZ), express them in the embryo, and if it's the right module/enhancer, then LacZ should be turned on in that stripe.What are segment polarity genes? What would the larval phenotype look like if the segment polarity genes are mutated? What regulates the expression of segment polarity genes? How is the boundary between the segments maintained?Segment polarity gene (gooseberry): responsible for the polarity of certain segments of the embryo (anterior or posterior) Regulative development can occur because we have CELLS, BOI. The different segments are established by... regulative development I think. Definitely cellular communication. ------- Engrailed is regulated by Eve and Ftz. Wingless induces the expression of Frizzled.What are homeotic genes? How are they turned on?They specify expression of a certain body part/anatomical structure. Homeotic genes are turned on with transcription factors They have homeodomains (DNA binding sites) They are spatially arranged in chromosomes according to the A-P position of the structure they specify.What would happen if you mis-expressed a homeotic gene in a different segment?You can cause formation of other body structures in the wrong place. Example, misexpression of eyeless (ey/pax-6) in these mutants leads to ectopic eyes.What would happen if you mutated a homeotic gene in a given segment?It would negatively affect anatomical formationAre homeotic genes just found in Drosophila? What are they called in mammals?No I believe they're called Hox genes.