Humbio 157

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LGR5

Back 1

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

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LacZ

Back 2

Reporter gene. Turns things blue.

Front 3

Rosa26

Back 3

The ubiquitous promoter

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DPP

Back 4

Ligand used in TGF-BEta pathway

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TGF-Beta

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Pathway that maintains (prevents diff) of GSCs

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Tamoxifen

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Steroid hormone used to activate CreER

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Schnurri (Shn)

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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

Front 8

Flp-FRT

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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

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Somites

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Produces blood bone and muscle

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Pax3

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Transcription Factor strongly associated with development of myogenic progenitors

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Quiescent

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

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Muscle Stem Cells

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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"

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Pax7

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Transcription factor for muscle stem cells

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Non-Canonical Wnt (Wnt7a)

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

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RBPJ

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

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Notch 3

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High at beginning of terminal diff of MSCs.

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Notch 1

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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

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Numb

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

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Beta-catenin

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

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APC

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

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Axin

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

Front 22

GSK3

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

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Germline stem cells

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

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DPP and Gbb

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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

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Bag of Marbles (BAM)

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

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Knockout of DPP experiment

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Shows necessity of TGFB signaling

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Enhancer Bashing

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

Front 28

Bone Stem Cells

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

Front 29

Somitogenesis

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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)

Front 30

Osterix (Osx)

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Marker of osteoprogenitor cells

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Myotome

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Makes muscle

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Schelerotome

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makes bone

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AER

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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

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Zone of Polarizing activity

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

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Sonic Hedgehog (Shh)

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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

Front 36

Hox genes

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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

Front 37

Chondrocytes

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

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Hypertrophy

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Enlargement and blood vessel recruitment

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Canonical Wnt

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

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Hematopoietic Stem cells

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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)

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

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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)

Front 42

Repopulating the Niche

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

Front 43

Parabiosis

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

Front 44

How to test if an effect is cell linked

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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

Front 45

Testing proliferation/differentiation of MSCs

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

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Driving hESCs towards a cell type

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

Front 47

BrdU

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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.

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Associated

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

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Cancer Stem Cells

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

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Reconstitution assay

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

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Transplantation assay

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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

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Neural Stem Cells

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

OR

->precursors ->Neuroblast->Neurons

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Two known sites of adult Neurogenesis

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

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NSCs and Cancer

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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)

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Epidermal Stem Cells

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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)

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Hair follicle SCs

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

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Skin Cell Cancer

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Basal cell carcinoma -> Hair follicle

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

Melanoma: Ras/Raf Ink4A

Front 58

Method for identifying cellular origin of different skin cancers

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

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Stem Cells and Aging

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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

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Cell-intrinsic "Irreversible" damage

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DNA damage, Telomere Erosion, Mitochondrial Dysfunction. Protein aggregates

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Insulin-Foxo pathway

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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

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ChiP Seq

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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

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Pioneering Factors

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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

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Endoderm

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Signaled by high Nodal for a long time

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Tetraploid Embryo Complementation

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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

Front 66

Bisulfate Sequencing

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Measures DNA Methylation

Front 67

DNA Fish

Back 67

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

Front 68

Reprogramming of pancreatic exocrine cells to beta cells

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Used Ngn3, Pdx1, and MAfA to convert

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microarray

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

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RNA Seq

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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.

Front 71

Blastema experiment

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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

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Reprogramming Methods

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