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Website reference
http://biology.kenyon.edu/courses/biol114/Chap14/Chapter_14.html
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Endoderm
the most internal germ layer, forms the lining of the gut and other internal organs.
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Ectoderm
the most exterior germ layer, forms skin, brain, the nervous system, and other external tissues
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Mesoderm
the the middle germ layer, forms muscle, the skeletal system, and the circulatory system
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What are the three processes of gastrulation?
- Patterning
- Cell fate specification
- Morphogenetic events
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Describe patterning.
- Dynamic, spatial and temporal specification of cell behaviors and tissue properties
- e.g. formation of limbs, spinal cord, somites, etc
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Describe cell fate specification
Determination (decision-making) of final fates
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Describe morphogenetic events
- Cell movements that result in unique morphological and functional structures
- e.g. neuron outside of spinal chord
- e.g. limb outside of somite
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What are the different types of cell movements during gastulation?
- Invagination
- Ingression
- Involution
- Intercalation
- Epiboly
- Convergent extension
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Invagination
Sheet of cells move inward
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Ingression
Single cells migrate as mesenchymal cells from epithelium
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Involution
- An epithelial group of cells rolls underneath to form a deeper level
- e.g. formation of embryonic cavity
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Intercalation
- Cells from two rows move between one other to form a single cell layer
- Results in increase in length (cell division and flattening)
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Epiboly
- Moving layer of cells on surface
- e.g. Epithelial cells spread across the embryo
- Most studied in zebrafish
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Convergent extension
- Highly directional intercalation.
- Cells converge by intercalating perpendicular to the axis of extension, resulting in the overall extension of the tissue in a preferred direction.
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Describe sea urchin gastrulation
- Cell adhesion properties of migrating cells change (driven by cadherin expression)
- Mesenchyme cells (multipotent embryonic connective tissue/cells) move by ingression and will form mesoderm
- Cells in vegetal plate undergo primary invagination to form archenteron
- Mouth forms at animal pole where archenteron meets ectoderm
- Vegetal pole forms anus
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What drives cell movements?
Cell adhesion properties of cadherins and integrins change binding to other cells and extracellular matrix
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Blastopore
- Region of embryo where cells move to begin forming mesoderm
- "Primitive streak" in mammals
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Describe mesenchyme
- Migrate by ingression
- Poorly differentiated
- Give rise to mesoderm
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What cell movements occur in Xenopus gastrulation?
- Invagination below center of gray crescent to form dorsal lip of future blastopore
- Animal pole cells move across surface and involute into interior of embryo to form endoderm and mesoderm
- Convergent extension when cells cover embryo and elongate
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What is a Bottle Cell?
Cells forming the blastapore lip that change shape (apical constriction) in frog gastrulation allowing cells to invaginate
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How does the blastopore lip of the frog form?
- Bottle cells change shape
- Hormones released which drive invagination
- Shape of cells allow migration between the cells (in theory)
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How would you show blastopore cells involved in invagination during gastrulation in frog?
- Cell ablation should result in lack of invagination
- Must also show hormones not produced by western or immunostaining
- Also can use transplantation to show that it occurs
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How would you show hormones are involved in invagination during gastrulation in frog?
Knockdown (not knockout since frog is not genetic organism) of hormone
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In frogs, the ___ cells move using ___ resulting in the ___ or ___ of the embryo.
involuting marginal zone (IMZ), convergent extension, lengthening, stretching
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In frogs, ___ cells ___ and instead of ___, their movement results in a ___. These cells produce ___ and ___.
ventral IMZ, intercalate, extending, thickening. ventral mesoderm, somites
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What mechanism of cell movement is involved in zebrafish gastrulation?
Epiboly
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What proteins are involved in zebrafish gastrulation?
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Describe Wnt in terms of zebrafish gastrulation.
- Both canonical (beta-catenin) and non-canonical pathways
- Regulates organizrs in both the frog and fish
- Regulates both cell movements and cell specification (differentiation)
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Describe Nodal in terms of zebrafish (all vertebrates) gastrulation.
- Axes formation
- mesoderm induction
- neural patterning
- left-right symmetry
- tissue morphogenesis
- Differentiation of mesodermal lineages
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Describe BMP in terms of zebrafish gastrulation.
- Dorso-ventral BMP gradient formed at beginning of gastrulation
- Mesoderm induction and dorso/ventral patterning of germ layers
- Modulates E-cadherin expression
- Promotes epibolic cell movements
- Cell prolifiation
- differentiation
- motility
- adhesion
- death
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Describe FGF in terms of zebrafish gastrulation.
Activates expression of Snail (transcription factor which inhibits E-cadherin transcription)
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What does FGF do in the chick?
Regulates cell movement inside embryo
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Describe FGF8 in the chick.
- Expressed in primitive streak
- Chemorepellant directing cells to move away from blastopore
- Cells move to blastopore and then release hormones to keep other cells away
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What does FGF8 do in the mouse?
high levels are required for normal mesodermal migration
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Describe FGF4 in the chick.
- Expressed in the extending axial mesoderm
- Chemoattractant for the dorsal convergence of the lateral mesoderm
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___ is expressed in the sphere stage of zebrafish, and is responsible for ___.
BMP, dorsoventral patterning and convergence and extension
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___ is expressed in the 50% ___ stage of zebrafish and is responsible for ___.
Wnt/beta-catenin, epiboly, dorsoventral patterning and germ layer separation
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___ is expressed in the shield stage of zebrafish and is responsible for ___.
FGF; dorsoventral patterning, germ layer separation, convergence and extension
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___ is expressed in the shield stage of zebrafish and is responsible for ___.
Nodal/TGF-beta; germ layer separation, convergence and extension
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___ is expressed in the 75% ___ stage of zebrafish and is responsible for ___.
Wnt/PCP and Wnt/PKC; germ layer separation, convergence and extension
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___ is expressed in the talibud stage of zebrafish and is responsible for ___.
GPCR; germ layer separation, convergence and extension
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The zebrafish embryo develops from ___.
the top
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In chick development, the ___ narrows and lengthens forming the ___ -- the chick's ___.
primitive streak, primitive groove, blastopore
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In chick development, ___ cells ___ at the midline and ___ at the primitive streak.
epiblast, converge, ingress
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What are epiblast cells?
In mammals, tissue derived from the inner cell mass at the end of cleavage
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What are trophoblast cells?
Form outer layer of blastocyst and develop into large part of the placenta
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Implantation
The embedding of the embryo intot he wall of the uterus
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In the human embryo, ___ cells develop first since the ___ and ___ are needed first.
anterior, brain, heart
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___ have a distinct pattern of ___ and ___.
Germ layer precursors, gene expression, morphogenetic movements
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Prospective ___ cells migrate as an ___ and go through ___.
ectodermal, epithelial layer, intercalation
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Prospective ___ cells migrate as ___ cells (loosely associated).
endodermal, mesenchymal
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Prospective ___ cells move as a ___ cell migration.
mesodermal, directed
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Prospective ___ cells exhibit ___ behavior.
endoderm, "random walk"
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What determines whether a cell becomes mesoderm or endoderm?
Physical location
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___ signalling is essential for ___ formation and ___ leading to that layer.
Nodal, mesoderm, cell movement
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___ modulation is critical for cell migration.
E-cadherin
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The canonical ___ pathway is involved in regulating ___ and ___.
Wnt, cell movements, cell specification
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The non-canonical ___ pathway regulates ___, but not directly to ___.
Wnt, morphogenetic movements, specification
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What are 5 common pathways used in early development and ruing patterning?
- Canonical Wnt
- Non-canonical Wnt
- TGF-beta/BMP
- Notch
- Hedgehog
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The 5 common pathways in early development are ___ in ___ and ___.
highly conserved, evolution, cancer
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For the Canonical Wnt pathway, in the absence of Wnt, ___.
beta-catenin is hyperphosphorylated
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How is beta-catenin hyperphosphorylated?
- Destroyed by "destruction" complex
- Ubiquitin binds
- Removed by proteosome
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For the Canonical Wnt pathway, when Wnt ligand binds to the receptor, ___, ___.
Frizzled/LRP-5/6, beta-catenin is stable (i.e. non-phosphorylated)
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What happens after the stabilization of beta-catenin?
- Translocated into the nucleus
- Interacts with TCF/LEF proteins
- Activates transcription
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Beta-catenin is a ___ and requires ___ to work. Its activation results in ___.
transcription factor, a complex of proteins. activation of cell division
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What is ubiquitin?
- Small molecule bound to proteins targetted for removal
- Results in gene repression
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For the non-canonical Wnt pathway, Wnt binds to ___.
- Frizzled (Fz) receptor, activating several transcription factors in the cytoplasm
- Neither LDL receptor nor beta-catenin are used
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Binding of Wnt in the non-canonical pathway leads to ___.
cell movements/migrations
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___ and ___ are not used in the non-canonical Wnt pathway but are used in the canonical Wnt pathway.
LRP receptor, beta-catenin
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The TGF-beta/BMP pathways lead to ___.
Differentiation
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TGF stands for ___ and is a ___.
Transforming Growth Factor, hormone
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TGF is a family of proteins that include ___ and is involved in ___.
Vg1, Nodal, BMPs (bone structures); patterning
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TGFs are ___ that ___ receptors on the membrame and activate ___ leading to ___.
dimers, cross-link, signal transduction pathways, differentiation
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SMADs activate ___.
differentiation genes
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Notch receptors are ___ composed of ___, ___, and ___ domains.
single-pass transmembrane proteins, functional extracellular (NECD), transmembrane, intracellular
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In mammals, members of the ___ and the ___ families, which are located in the ___ cell, function as ___ that activate ___.
Delta-like, Jagged, signal-sending, ligands, Notch signaling receptors
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Upon ligand binding, the ___ is cleaved away (___ cleavage) from the ___ domain by ___.
NECD, S2, TM-NICD, TACE (ADAM metalloprotease TNF-alpha converting enzyme)
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After cleavage, the ___ remains bound to the ligand and this complex undergoes ___ and ___ within the ___.
NECD, endocytosis, recycling/degradation, signal-sending cell
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In the signal-receiving cell, a ___ event mediated by ___ releases the ___ from the ___ (___).
third cleavage, gamma-secretase, NICD, TM, S3 cleavage
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After the third cleavage, ___ translocates to the ___ and associates with the ___ family transcription factor complex which results in ___.
NICD, nucleus, CSL (CBF1/Su(H)/Lag-1), activation of the notch target genes
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Question for Dr. Soto: In notch pathway, what are the three cleavage events?
- Intra and extra cellular domains cleave
- TM from NICD
- ?
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What occurs after NICD enters the nucleus?
- Activation of transcription
- Cell division
- Differentiation
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___ or ___ can bind to the notch receptor.
Delta, Jagged
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Describe the Hedgehog pathway.
- Contains a family of secreted proteins (hormones)
- Found in vertebrates and invertebrates
- Function in development
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What ligands are involved in the Hedgehog pathway?
- Invertebrates: hedgehog
- Vertebrates: sonic, desert, Indian hedgehob
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What receptors are involved in the hedgehob pathway?
- both: Patch
- invertebrates: smoothened
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What is the ultimate target of the Hedgehog pathway in the fruitfly?
cubitus interruptus (Ci), a transcription factor
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What is the role in development of the Hedgehog pathway?
Cellular proliferation, growth, axon path finding, and somite development in vertebrates
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Describe Patch.
- Ptc is a membrane receptor that binds to Hh to activate Smo
- Levels of membrane Ptc decrease after binding due to endocytosis
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Describe Smoothened.
Smo is an intermembrane protein that, when activated, relays signals to HSC
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Describe the Intracellular Hedgehog Signalling Complex (HSC)
- Coastal 2 (Co2): kinesin-related protein
- Fused (Fu): Ser/Threo kinase
- Supressor of fused (Su/Fu)
- Ci
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What happens if there is no Hh?
HSC is attached to microtubule (MT) complex which truncates Ci which becomes a repressor
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What happens when Hh is present?
Differential binding of Hh to PTC leads to dissociation from MT complex and production of Ci trans-activators which activates target genes
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It is not understood how ___ goes from being ___ to soluble when Hh binds.
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Hh proteins are not released ___; they are released into ___.
freely; cytoneme (long and thin tubes connecting cells)
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