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the most internal germ layer, forms the lining of the gut and other internal organs.
the most exterior germ layer, forms skin, brain, the nervous system, and other external tissues
the the middle germ layer, forms muscle, the skeletal system, and the circulatory system
What are the three processes of gastrulation?
- Cell fate specification
- Morphogenetic events
- Dynamic, spatial and temporal specification of cell behaviors and tissue properties
- e.g. formation of limbs, spinal cord, somites, etc
Describe cell fate specification
Determination (decision-making) of final fates
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
What are the different types of cell movements during gastulation?
- Convergent extension
Sheet of cells move inward
Single cells migrate as mesenchymal cells from epithelium
- An epithelial group of cells rolls underneath to form a deeper level
- e.g. formation of embryonic cavity
- Cells from two rows move between one other to form a single cell layer
- Results in increase in length (cell division and flattening)
- Moving layer of cells on surface
- e.g. Epithelial cells spread across the embryo
- Most studied in zebrafish
- 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.
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
What drives cell movements?
Cell adhesion properties of cadherins and integrins change binding to other cells and extracellular matrix
- Region of embryo where cells move to begin forming mesoderm
- "Primitive streak" in mammals
- Migrate by ingression
- Poorly differentiated
- Give rise to mesoderm
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
What is a Bottle Cell?
Cells forming the blastapore lip that change shape (apical constriction) in frog gastrulation allowing cells to invaginate
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)
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
How would you show hormones are involved in invagination during gastrulation in frog?
Knockdown (not knockout since frog is not genetic organism) of hormone
In frogs, the ___ cells move using ___ resulting in the ___ or ___ of the embryo.
involuting marginal zone (IMZ), convergent extension, lengthening, stretching
In frogs, ___ cells ___ and instead of ___, their movement results in a ___. These cells produce ___ and ___.
ventral IMZ, intercalate, extending, thickening. ventral mesoderm, somites
What mechanism of cell movement is involved in zebrafish gastrulation?
What proteins are involved in zebrafish gastrulation?
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)
Describe Nodal in terms of zebrafish (all vertebrates) gastrulation.
- Axes formation
- mesoderm induction
- neural patterning
- left-right symmetry
- tissue morphogenesis
- Differentiation of mesodermal lineages
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
Describe FGF in terms of zebrafish gastrulation.
Activates expression of Snail (transcription factor which inhibits E-cadherin transcription)
What does FGF do in the chick?
Regulates cell movement inside embryo
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
What does FGF8 do in the mouse?
high levels are required for normal mesodermal migration
Describe FGF4 in the chick.
- Expressed in the extending axial mesoderm
- Chemoattractant for the dorsal convergence of the lateral mesoderm
___ is expressed in the sphere stage of zebrafish, and is responsible for ___.
BMP, dorsoventral patterning and convergence and extension
___ is expressed in the 50% ___ stage of zebrafish and is responsible for ___.
Wnt/beta-catenin, epiboly, dorsoventral patterning and germ layer separation
___ is expressed in the shield stage of zebrafish and is responsible for ___.
FGF; dorsoventral patterning, germ layer separation, convergence and extension
___ is expressed in the shield stage of zebrafish and is responsible for ___.
Nodal/TGF-beta; germ layer separation, convergence and extension
___ is expressed in the 75% ___ stage of zebrafish and is responsible for ___.
Wnt/PCP and Wnt/PKC; germ layer separation, convergence and extension
___ is expressed in the talibud stage of zebrafish and is responsible for ___.
GPCR; germ layer separation, convergence and extension
The zebrafish embryo develops from ___.
In chick development, the ___ narrows and lengthens forming the ___ -- the chick's ___.
primitive streak, primitive groove, blastopore
In chick development, ___ cells ___ at the midline and ___ at the primitive streak.
epiblast, converge, ingress
What are epiblast cells?
In mammals, tissue derived from the inner cell mass at the end of cleavage
What are trophoblast cells?
Form outer layer of blastocyst and develop into large part of the placenta
The embedding of the embryo intot he wall of the uterus
In the human embryo, ___ cells develop first since the ___ and ___ are needed first.
anterior, brain, heart
___ have a distinct pattern of ___ and ___.
Germ layer precursors, gene expression, morphogenetic movements
Prospective ___ cells migrate as an ___ and go through ___.
ectodermal, epithelial layer, intercalation
Prospective ___ cells migrate as ___ cells (loosely associated).
Prospective ___ cells move as a ___ cell migration.
Prospective ___ cells exhibit ___ behavior.
endoderm, "random walk"
What determines whether a cell becomes mesoderm or endoderm?
___ signalling is essential for ___ formation and ___ leading to that layer.
Nodal, mesoderm, cell movement
___ modulation is critical for cell migration.
The canonical ___ pathway is involved in regulating ___ and ___.
Wnt, cell movements, cell specification
The non-canonical ___ pathway regulates ___, but not directly to ___.
Wnt, morphogenetic movements, specification
What are 5 common pathways used in early development and ruing patterning?
- Canonical Wnt
- Non-canonical Wnt
The 5 common pathways in early development are ___ in ___ and ___.
highly conserved, evolution, cancer
For the Canonical Wnt pathway, in the absence of Wnt, ___.
beta-catenin is hyperphosphorylated
How is beta-catenin hyperphosphorylated?
- Destroyed by "destruction" complex
- Ubiquitin binds
- Removed by proteosome
For the Canonical Wnt pathway, when Wnt ligand binds to the receptor, ___, ___.
Frizzled/LRP-5/6, beta-catenin is stable (i.e. non-phosphorylated)
What happens after the stabilization of beta-catenin?
- Translocated into the nucleus
- Interacts with TCF/LEF proteins
- Activates transcription
Beta-catenin is a ___ and requires ___ to work. Its activation results in ___.
transcription factor, a complex of proteins. activation of cell division
What is ubiquitin?
- Small molecule bound to proteins targetted for removal
- Results in gene repression
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
Binding of Wnt in the non-canonical pathway leads to ___.
___ and ___ are not used in the non-canonical Wnt pathway but are used in the canonical Wnt pathway.
LRP receptor, beta-catenin
The TGF-beta/BMP pathways lead to ___.
TGF stands for ___ and is a ___.
Transforming Growth Factor, hormone
TGF is a family of proteins that include ___ and is involved in ___.
Vg1, Nodal, BMPs (bone structures); patterning
TGFs are ___ that ___ receptors on the membrame and activate ___ leading to ___.
dimers, cross-link, signal transduction pathways, differentiation
SMADs activate ___.
Notch receptors are ___ composed of ___, ___, and ___ domains.
single-pass transmembrane proteins, functional extracellular (NECD), transmembrane, intracellular
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
Upon ligand binding, the ___ is cleaved away (___ cleavage) from the ___ domain by ___.
NECD, S2, TM-NICD, TACE (ADAM metalloprotease TNF-alpha converting enzyme)
After cleavage, the ___ remains bound to the ligand and this complex undergoes ___ and ___ within the ___.
NECD, endocytosis, recycling/degradation, signal-sending cell
In the signal-receiving cell, a ___ event mediated by ___ releases the ___ from the ___ (___).
third cleavage, gamma-secretase, NICD, TM, S3 cleavage
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
Question for Dr. Soto: In notch pathway, what are the three cleavage events?
- Intra and extra cellular domains cleave
- TM from NICD
What occurs after NICD enters the nucleus?
- Activation of transcription
- Cell division
___ or ___ can bind to the notch receptor.
Describe the Hedgehog pathway.
- Contains a family of secreted proteins (hormones)
- Found in vertebrates and invertebrates
- Function in development
What ligands are involved in the Hedgehog pathway?
- Invertebrates: hedgehog
- Vertebrates: sonic, desert, Indian hedgehob
What receptors are involved in the hedgehob pathway?
- both: Patch
- invertebrates: smoothened
What is the ultimate target of the Hedgehog pathway in the fruitfly?
cubitus interruptus (Ci), a transcription factor
What is the role in development of the Hedgehog pathway?
Cellular proliferation, growth, axon path finding, and somite development in vertebrates
- Ptc is a membrane receptor that binds to Hh to activate Smo
- Levels of membrane Ptc decrease after binding due to endocytosis
Smo is an intermembrane protein that, when activated, relays signals to HSC
Describe the Intracellular Hedgehog Signalling Complex (HSC)
- Coastal 2 (Co2): kinesin-related protein
- Fused (Fu): Ser/Threo kinase
- Supressor of fused (Su/Fu)
What happens if there is no Hh?
HSC is attached to microtubule (MT) complex which truncates Ci which becomes a repressor
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
It is not understood how ___ goes from being ___ to soluble when Hh binds.
Hh proteins are not released ___; they are released into ___.
freely; cytoneme (long and thin tubes connecting cells)