Morphogenesis + Stem Cells (lec 6/7/8) Flashcards
(T/F) Ribosomes have the same proteins on all tissues.
False!
Ribosomes have slightly different proteins depending on the tissue in which they reside in!
Ribosomal protein _______ is concentrated in those ribosomes found in the _______ that give rise to the vertebrae.
A wild-type embryo has normal vertebrae and normal Hox gene translation. Mice deficient in the protein have _______ hox gene translation and ______ ______ of vertebrae.
Rpl38 (protein 38 of the large ribosomal subunit); Somites
reduced; extra pair
(T/F) dsRNA and miRNAs are used for RNA interference. dsRNA are added to a cell and miRNAs are produced through transcription.
True!
miRNAs are small transcripts that do not encode for proteins, and parts of it can form hairpin, secondary structures on itself, yielding a dsRNA.
miRNAs target mRNA, leading to its degradation and not being translated - less protein made!
miRNAs are processed by the _____ _____ in the cell and then exported into the _________ where it will interact with the ____-_____ ________ _______, made up primarily of _________ and __________, that prepares the RNA to be used as a guide for targeted mechanisms of interference.
Drosha RNAase; Cytoplasm; RNA-induced silencing complex (RISC); Dicer; Argonaute
What is the role of miR430 during the maternal-to-zygotic transition in zebrafish?
Numerous mRNAs derived from maternal contributions fuel development during the cleavage stages, but transitioning into the gastrula requires active transcription of the zygotic genome.
miRNAs play a major role in CLEARING THESE MATERNALLY DERIVED TRANSCRIPTS during this transition.
miR430 plays a major role in the interference of a majority of maternal transcripts in the zebrafish blastula as it transitions to zygotic control during gastrulation.
Match the different types of localization of mRNAs in the oocyte to their definitions:
1) Diffusion and local anchoring
2) Localized protection
3) Active transport along cytoskeleton
A) bicoid mRNA is TRANSPORTED ALONG MICROTUBULES by the motor protein DYNEIN to the anterior of the oocyte. Oskar mRNA is brought to the posterior by the motor protein KINESIN along microtubules.
B) nanos mRNA DIFFUSES through the egg and is BOUND (in part by the Oskar protein) at the POSTERIOR end of the oocyte. This anchoring allows the mRNA to be translated.
C) the mRNA for heat shock protein (Hsp83) will be DEGRADED UNLESS IT BINDS to a PROTECTOR PROTEIN (also at the POSTERIOR end)
Diffusion and local anchoring: nanos mRNA DIFFUSES through the egg and is BOUND (in part by the Oskar protein) at the POSTERIOR end of the oocyte. This anchoring allows the mRNA to be translated.
Localized protection: the mRNA for heat shock protein (Hsp83) will be DEGRADED UNLESS IT BINDS to a PROTECTOR PROTEIN (also at the POSTERIOR end)
Active transport along cytoskeleton: bicoid mRNA is TRANSPORTED ALONG MICROTUBULES by the motor protein DYNEIN to the anterior of the oocyte. Oskar mRNA is brought to the posterior by the motor protein KINESIN along microtubules.
In _____ __________ is a very important method to visualize where mRNAs are located.
For whole mount in situ hybridization for ODD-SKIPPED mRNA in a stage-9 drosophila embryo, antisense RNA probe with _______ _______ is conjugated to ___________.
The probe with complementarity to the odd-skipped gene becomes __________ to any cell expressing oddskipped transcripts.
Then samples are treated with ______ antibodies conjugated to the enzyme ________ __________.
This enzyme converts _______ and ________ to a blue precipitate. Thus , cells expressing the ODD-SKIPPED turn blue!
Situ hybridization
uridine triphosphate; digoxigenin (DIG)
hybridized
Anti-DIG; Alkaline phosphatase
NBT (Nitroblue tetrazolium chloride) and BICIP (5-bromo-4-chloro-3-indolyl-phosphate).
Briefly describe chromatin immunoprecipitation sequencing (ChIP-Seq) and how it works.
ChIP-Seq is another way to visualize the genome in development context.
Chromatin is isolated from the cell nuclei.
The chromatin proteins are crosslinked to their DNA-binding sites and the DNA is fragmented into small pieces.
Antibodies bind to specific chromatin proteins and the antibodies (and whatever bound to them) are precipitated out of the solution.
The DNA fragments associated with the precipitated complexes are purified from the proteins and sequenced.
These sequences can be compared with the genome maps to discover the precise locations of the genes these proteins may be regulating.
(T/F) Using RNA sequencing, you can sequence the entire genome or isolate tissues at different times and see which genes are being activated.
True!
Briefly describe how RNA sequencing works in 4 steps.
1) RNA is isolated to obtain only those genes that are actively expressed
2) These transcripts are then fragmented into smaller stretches and used to create cDNA with reverse transcriptase
3) Specialized adaptors are ligated to the cDNA ends to enable PCR amplification and immobilization for:
4) Subsequent sequencing
What is the CRISPR/Cas9 system used for?
It is used to cause targeted indel formation or insertional mutagenesis within a gene of interest.
Briefly fill in the blanks regarding the CRISPR/Cas 9 system:
A _____ ______ ____ RNA is designed and introduced into cells together with the nuclease _____, for instance by co-injection into a newly fertilized zygote.
That RNA will bind to the genome with _______ and will recruit the nuclease to induce a _____-______ break, which is fixed by NHEJ.
Gene-specific guide RNA (gRNA); Cas9
Complementarity; Double-stranded
What is NHEJ?
Non-homologous end joining is the cell’s DNA repair mechanism that often results in small INSERTIONS or DELETIONS (~2-30 base pairs), which can cause the establishment of a premature stop codon and potential loss of the protein’s function.
(T/F) Plasmids carrying insertions with homology to regions surrounding the gRNA target sites are used to insert known sequences at the double stranded break induced by Cas9. Such methods are being explored as a way to repair mutations.
True!
Briefly describe the targeted expression of the Pax6 gene in the mouth using the GAL4 system in drosophila.
The gene for the GAL4 transcription factor was placed downstream from an enhancer sequence that normally stimulates gene expression for imaginal discs for mouthparts.
If crosses to a strain that has a transgene that places GAL4-binding sites upstream of the Pax6 gene, IN THE PROGENY, the Pax6 gene will be expressed in whichever the imaginal disc the GAL4 protein is made.
In this case, Pax6 gene was expressed in the mouth part discs.
Briefly describe the Cre-lox technique for conditional mutagenesis.
The Cre-lox induces gene mutations in SPECIFIC CELLS ONLY!
For example, mice wild-type alleles (for any gene) were REPLACED BY ALLELES in which the second exon was FLANKED BY loxP sequences.
Then, they were MATED with mice having the gene for CRE-RECOMBINASE fused to a PROMOTER ACTIVE ONLY IN PARTICULAR CELLS (example: promoter is of albumin gene that functions only in liver).
In mice with BOTH of the altered alleles (gene flanked by loxP and gene for Cre-recombinase with a promoter), Cre-recombinase is made only in cells where the promoter is activated (liver cells that synthesize albumin).
The Cre-recombinase binds to the loxP sequences flanking exon 2 and REMOVES THE EXON. Then, only the liver cells lack the functional gene!
Match the different types of signaling to their definitions:
1) Juxtracrine signaling
2) Paracrine signaling
3) Endocrine signaling
4) Autocrine signaling
A) Signaling ACROSS MULTIPLE CELL DISTANCES, whereby one cell secretes a signaling protein (LIGAND) into the environment and across the distance of many cells. Only those cells expressing this ligand’s RECEPTOR can response, EITHER RAPIDLY through chemical reactions in the cytosol OR MORE SLOWLY through the process of gene expression.
B) Long range glandular/inter organ signaling of hormones.
C) A cell signaling to itself.
D) LOCAL CELL SIGNALING is carried out VIA MEMBRANE RECEPTORS that bind to proteins in the extracellular matrix (ECM) or directly to receptors from a neighbouring cell.
Juxtracrine signaling: LOCAL CELL SIGNALING is carried out VIA MEMBRANE RECEPTORS that bind to proteins in the extracellular matrix (ECM) or directly to receptors from a neighbouring cell.
Paracrine signaling: Signaling across MULTIPLE CELL DISTANCES, whereby one cell secretes a signaling protein (LIGAND) into the environment and across the distance of many cells. Only those cells expressing this ligand’s RECEPTOR can response, EITHER RAPIDLY through chemical reactions in the cytosol OR MORE SLOWLY through the process of gene expression.
Endocrine signaling: Long range glandular/inter organ signaling of hormones.
Autocrine signaling: A cell signaling to itself.
What would happen if presumptive epidermal cells and neural plate cells were dissociated and mixed together?
Why?
The cells REAGGREGATE so one type (presumptive epidermis) covers the other.
The cells with a GREATER cell COHESION segregate inside the cells with LESS COHESION.
(T/F) Because limb buds have a greater surface tension than epithelium, they re-organize themselves inside the epithelium.
True!
Aggregate surface tension correlates with the number of _________ molecules on the cell membranes.
cadherin
*if red cells have more cadherin than green cells, then red cells sort to the center with green cells at periphery.
(T/F) There are three different types of cadherin molecules (N-cadherin, P-cadherin, and E-cadherin). Sorting of cells can occur based on cadherin number even if the two cells express different cadherin proteins.
True!
E-cadherin is required for ______ in zebrafish.
During normal gastrulation, cells merge into a ______ but more expansive ________ layer that envelopes the entire _______.
E-cadherin mutants fail to complete ________, which is most severely impaired in the _________ mutant.
epiboly
thinner; epiblast; yolk
epiboly; homozygous
(T/F) During the formation of epiboly in zebrafish, cells move toward the superficial enveloping layer in relationship to decreasing expression of E-cadherin.
False!
During the formation of epiboly in zebrafish, cells move toward the superficial enveloping layer in relationship to INCREASING expression of E-cadherin.
E-cadherin is expressed at higher levels in the more superficial layers of the epiblast, including the envelope layer, and it is this differential expression (/adhesion) that POWERS the RADICAL MOVEMENT of deep cells to the PERIPHERY!
The ________ orients the movements of the mesodermal cells.
fibronectin
What are integrins?
How do they achieve their function?
Integrins function as TRANSMEMBRANE LINKERS (or “integrators”), mediating the interactions between the CYTOSKELETON and the EXTRACELLULAR MATRIX that are required for the cells to grip the matrix.
They form HETERODIMERIZED membrane-spanning receptor proteins that bind FIBRONECTIN on the outside of the cell while interacting with actin cytoskeleton-associated proteins on the INSIDE OF THE CELL.
Give examples of cytoskeleton associated proteins found in the inside of cells.
α-Actinin, Vinculin, and Talin
(T/F) Integrin is composed of three different types do subunits (α, β, and γ).
False! It is composed of TWO different types do subunits (α and β).
Normal epithelial cells are attached to one another through adherens junctions containing _______, _____, and _____ rings.
They are attached to the _______ lamina (? matrix) through ________.
_________ factors can repress the expression of genes that encode these cellular components, causing the cell to lose ______, lose _______ to the basal lamina, and lose ______ with other epithelial cells.
Cadherin; catenins; and actin
Basal (extracellular matrix); integrins
Paracrine; polarity; attachement; cohesion
Describe the epithelial-mesenchymal transition (EMT).
After epithelial cells lose polarity, attachment to basal lamina and cohesion to each other due to paracrine factors.
CYTOSKELETAL REMODELLING occurs, as well as the SECRETION OF PROTEASES that degrade the basal lamina and other extracellular matrix components of the basement membrane, ENABLING THE MIGRATION OF THE NEWLY FORMED MESENCHYMAL CELL.
