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72 Cards in this Set

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LGR5

Target gene of Wnt, expressed in cycling crypt base columnar cells. Can be used to detect Wnt up or down regulation

LacZ

Reporter gene. Turns things blue.

Rosa26

The ubiquitous promoter

DPP

Ligand used in TGF-BEta pathway

TGF-Beta

Pathway that maintains (prevents diff) of GSCs

Tamoxifen

Steroid hormone used to activate CreER

Schnurri (Shn)

Critical repressive transcriptional factor in the TGF-B pathway. When Wnt pathway is activated, MAD binds to Shn to turn it into a repressive complex, preventing differentiation

Flp-FRT

Used to create homozygous mutant cells for a particular gene. Used when a homozygous embryo would not be viable. Happens during mitosis after duplication. At the end, the homozygous cells will be the non labeled (non blue if its LacZ) ones

Somites

Produces blood bone and muscle

Pax3

Transcription Factor strongly associated with development of myogenic progenitors

Quiescent

Not always active in cell cycle. Maybeneeds to be injured before activating (muscle sc) vs Cycling (intestine,germline)

Muscle Stem Cells

Satellite cells. Express Pax7, mononucleated. Important signals include Wnt (noncanon and canonical), Notch3, Notch 1. Niche of MSCs are the muscle fibers themselves (producing Delta) Repairs muscle damage. Quiescence must be actively maintained (promoted by Notch), but cells are "poised"

Pax7

Transcription factor for muscle stem cells

Non-Canonical Wnt (Wnt7a)

Does not involve Beta Catenin. Leads to expansion of stem cell pop (Self renewal) by regulating plane of division (creates gradient?)

RBPJ

Transcription factor downstream of Notch3. Knockout experiment showed that Notch3 is required for SC maintenance

Notch 3

High at beginning of terminal diff of MSCs.

Notch 1

Starts low, high during transit amplifying state, lowers to complete differentiation. Is inhibited by Numb. Must lower by end because Notch activates GSK3B, an inhibitor of canonical Wnt

Numb

Inhibits Notch. Low levels promote transit amplifying stage. High levels promote further differentiation by inhibiting Notch to promote canonical Wnt signaling

Beta-catenin

Transcription factor that binds to DNA as the result of the Wnt pathway

APC

In Wnt pathway. Destroys Beta Catenin. Mutations of APC linked to cancer of the colon

Axin

In Wnt pathway. Destroys Beta Catenin. Mutations of Axin linked to cancer of the liver

GSK3

In Wnt pathway. Works with APC and Axin to destroy Beta Catenin

Germline stem cells

Study of drosophila ovaries. Has 2 GSCs. Uses Transit Amplifying stage. Eventually creates a single oocyte, several nurse cells

DPP and Gbb

BMPs that are signalers in TGFB pathway. Activates MAD which represses BAM via Shn. BAM usually promotes differentiation. Repression of BAM helps maintain stem cells. Dpp is sufficient for self-renewal

Bag of Marbles (BAM)

Promotes differentiation. Mutants of BAM get a ton of stem cells that don't differentiate and apparently look like a bag of marbles.

Knockout of DPP experiment

Shows necessity of TGFB signaling

Enhancer Bashing

When you remove a sequence upstream of a gene that you suspect to be regulatory. Used to create Drosophila GSC model

Bone Stem Cells

Come from mesenchymal stem cells. Osteoprogenitors are used to mark the bone lineage. Important regulators of Wnt/FGF, Retinoic Acid,

Somitogenesis

Gradient of FGF/Wnt (posterior) and Retinoic Acid (anterior) help to regulate somitogenesis clock, resulting in formation of a somite every two hours in the mouse model. This is for the axial skeleton (spine)

Osterix (Osx)

Marker of osteoprogenitor cells

Myotome

Makes muscle

Schelerotome

makes bone

AER

Ridge on the outside of the limb bud that sends signals that regulates proximal-distal growth. When removed at different times in development, different parts of the limb fail to form - shows similar periodic dev process

Zone of Polarizing activity

Regulates anterior-posterior patterning with help of sonic hedgehog (shh)

Sonic Hedgehog (Shh)

If you add a bead of retinoic acid (which induces Shh) to a limb bud, will induce a set of digits on the other side, showing sufficiency

Hox genes

Set of genes that controls vertebrae type. Ex: if you lose Hox 10 the lumbar turns to thoracic. If you gain Hox10 the thoracic goes to lumbar. The number of the Hox gene corresponds to the part of the vertebrae in a parallel sequence

Chondrocytes

Are like bone precursors. Moving from proliferation to hypertropy regulated by balance between PTHrP and lhh

Hypertrophy

Enlargement and blood vessel recruitment

Canonical Wnt

Required for osteoblast commitment. Seen in experiment where Beta catenin was destroyed. Could not form bone

Hematopoietic Stem cells

Generates all blood cell types. Can get HSCs from bone marrow, cord blood, and mobilized peripheral blood. Isolate via FACs sorting to find different types (multipotencty)

Experiment to test for if Wnt signaling contributes to production of particular cells.

Take LGR5-CreER mice and add tamoxifen to mark cells with Wnt activation. Next perform lineage tracing with the R26-loxP-STOP-loxP-LacZ assay and stain for LGR5 and also the antibodies of the proteins specific to the cell type that you're looking for (albumin for hepatocytes, CK19 for bile ducts)

Repopulating the Niche

If any SCs remain can be done by symmetric division. If not, transit amplifying cells may de-differentiate

Parabiosis

Experiment that pairs mice heterochronically and Isochronically to share a circulatory system. Used to test how circulating regulatory factors affect regenerative capacity.

How to test if an effect is cell linked

Usually done to specify results of Parabiosis. Take blood of young and old mice and purify it (removing the cells). Inject serum from young mice into old mice and compare this with injecting serum from old mouse into old mouse and results of parabiosis

Testing proliferation/differentiation of MSCs

Measured by incorporating BrdU or any nucleotide analogue. Also pH3 and Ki67 staining. Co-stain for differentiation markers like MyoD

Driving hESCs towards a cell type

Try to use methods (like supplementing inhibitor/activators in culture media) that don't manipulate hESC genome by integration

BrdU

Marker often used for label retention or measuring proliferation. Label retention is more useful for actively cycling cells (ie intestine) versus quiescent (muscle sc) because labels are retained in cells when incorporated into replicating DNA strands. This can lead to confusion with TA cells if they divide at a similar rate.

Associated

Term used when a factor is related to some effect or process but maybe not necessary or sufficient

Cancer Stem Cells

Much the of the same properties as other stem cells (markers, isolation techniques). Have been found in Breast, Brain, colon cancer and lymphomas

Reconstitution assay

Ex for hematopoietic stem cells: Removal all blood cell type progenitors, see if transplant can repopulate. This demonstrates multipotency

Transplantation assay

ex for HSCs: Removal all blood stem cells from a mouse, transplant a few in and see if they repopulate. Then take these newly generated SCs and perform a secondary transplant. If it repopulates, this is evidence of self-renewal

Neural Stem Cells

->precursors ->Glial prog -> Astrocyte or Oligodendrocyte


OR


->precursors ->Neuroblast->Neurons

Two known sites of adult Neurogenesis

Subventricular zone (olfactory bulb) and Hippocampal denate gyrus (Granule cell neurons)

NSCs and Cancer

Neural progs are highly vulnerable to death by irradiation. New treatment strategy is to introduce new NPCs into brain via transplantation. Can be via isograft (from same person) or allograft (from different person)

Epidermal Stem Cells

5 compartments: Interfollicular epidermis, hair follicle, sebaceous gland, sweat gland, touch dome. Each autonomously maintained by different stem cell pool, though there is some exchange in the context of injury (ex: hair follicle contributing to interfollicular epidermis)

Hair follicle SCs

Sit in bulge. Markers include K15, K14, CD34. Contributes to epidermis during wounding, but not really otherwise. Hedgehog is essential for hair follicle formation

Skin Cell Cancer

Basal cell carcinoma -> Hair follicle


Squamous cell carcinoma->Ras activation (ex. KRas)


Melanoma: Ras/Raf Ink4A

Method for identifying cellular origin of different skin cancers

Introduce mutations (Ex: For basal cell carcinoma, activate Hedgehog signaling) in candidate skin compartments respectively (one by one) to see which induces cancer

Stem Cells and Aging

Depending on the lineage, the number of SCs may increase (ex: HSCs) or decrease (NSCs) but potency declines with age universally. May experience skewing to one adult type (ex Glial rather than neurons for HSCs). Can study with Parabiosis

Cell-intrinsic "Irreversible" damage

DNA damage, Telomere Erosion, Mitochondrial Dysfunction. Protein aggregates

Insulin-Foxo pathway

FOXO promotes genes for longevity (Telomere length), but is usually inhibited by Insulin/IGF-I. Loss of FOXO will result in skewing towards Glial lineage

ChiP Seq

Tags with antibodies that recognize specific chromatin proteins. 1) Fragment the chromatin 2) Add antibodies 3) Purify immunocomplexes 4) Isolate DNA 5) High thoroughput sequencing 6) Align and count. Able to measure histone modification

Pioneering Factors

Factors that are able to bind to DNA that is wrapped around chromatin. Active Role: Opening chromatin to allow other factors to bind. Passive Role: Binds to location which recruits other factors to bind there

Endoderm

Signaled by high Nodal for a long time

Tetraploid Embryo Complementation

Fusion of 2N cells to form 4N cells in a embryo. Insert donor iPS cells. 4N cells die off, leaving the donor cells to form embryo. If you get a full iPSC derived mouse, you have proved that every cell of the embryo can be derived from the iPSc line

Bisulfate Sequencing

Measures DNA Methylation

DNA Fish

Looks at Chromatin structure - shows you where a chromosome is in DNA, but is fairly low resolution

Reprogramming of pancreatic exocrine cells to beta cells

Used Ngn3, Pdx1, and MAfA to convert

microarray

Fluorescently label one data set with green, another red. Flow these onto the array and measure relative fluorescence. Indicates gene expression

RNA Seq

Isolate RNA from induced neurons and neurons from a mouse. Convert RNA to cDNA. Barcode each sample and sequence cDNA. Map the sequenced reads to the genomes and identify differences in expression based on number of reads.

Blastema experiment

Dissected/transplanted cells of one germ layer from a GFP+ embryo in to adult with amputated arm. GFP+ cells only contributed to one layer (mesoderm). Shows that cells are committed to their germ layer

Reprogramming Methods

Nuclear Transfer or cell fusion. Ex: Forced expression of MyoD in fibroblasts lead to a muscle cell